Edisi 70 Medical Book

Volume 70Number 8 - August/2015

CLINICSEditor

Edmund Chada BaracatFaculdade de Medicina da Universidade de São Paulo

São Paulo, SP, Brazil

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Ana Maria de Ulhoa EscobarFaculdade de Medicina da Universidade de São PauloSão Paulo, SP, Brazil

Anna Sara Shafferman LevinFaculdade de Medicina da Universidade de São PauloSao Paulo, SP, Brazil

Antonio Egidio NardiUniversidade Federal do Rio de JaneiroRio de Janeiro, RJ, Brazil

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Clinics. São Paulo: Scientific Journal of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, 2005-

Monthly Periodical: January to December

ISSN 1807-5932 printed version

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ISSN-1807-5932

CLINICS

CONTENTS

Clinics 2015 70(8):535–599

CLINICAL SCIENCES

The negative prognostic impact of bone metastasis with a tumor massBirsen Yücel, Mustafa Gürol Celasun, Bilge Öztoprak, Zekiye Hasbek, Seher Bahar, Turgut Kacan,Aykut Bahceci, Mehmet Metin Seker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535

Ocular risk management in patients undergoing general anesthesia: an analysis of 39,431 surgeriesNewton Kara-Junior, Rodrigo Franca de Espindola, Joao Valverde Filho, Christiane Pellegrino Rosa,Andre Ottoboni, Enis Donizete Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541

Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscletransplantation in brachial plexus injuryYi Hou, Jiantao Yang, Yi Yang, Bengang Qin, Guo Fu, Xiangming Li, Liqiang Gu, Xiaolin Liu,Qingtang Zhu, Jian Qi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544

Treatment with dasatinib or nilotinib in chronic myeloid leukemia patients who failed to respond to twopreviously administered tyrosine kinase inhibitors – a single center experienceBeatriz Felicio Ribeiro, Eliana C.M. Miranda, Dulcinéia Martins de Albuquerque, Márcia T. Delamain,Gislaine Oliveira-Duarte, Maria Helena Almeida, Bruna Vergílio, Rosana Antunes da Silveira,Vagner Oliveira-Duarte, Irene Lorand-Metze, Carmino A. De Souza, Katia B.B. Pagnano . . . . . . . . . . . . . . . . . . . . 550

The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanismKun Zou, Caigang Liu, Zhuo Zhang, Lijuan Zou . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556

Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-year retrospective studyJi Xiong, Jun Wang, Juan Huang, Wenjing Sun, Jun Wang, Dongfeng Chen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis inumbilical vein endothelial cellsSher Zaman Safi, Kalaivani Batumalaie, Marzida Mansor, Karuthan Chinna, Syam Mohan,Selva Kumar, Hamed Karimian, Rajes Qvist, Muhammad Aqeel Ashraf, Garcie Ong Siok Yan . . . . . . . . . . . . . . . . 569

BASIC RESEARCH

Effect of hypertonic saline treatment on the inflammatory response after hydrochloric acid-inducedlung injury in pigsCarla Augusto Holms, Denise Aya Otsuki, Marcia Kahvegian, Cristina Oliveira Massoco, Denise Tabacchi Fantoni,Paulo Sampaio Gutierrez, Jose Otavio Costa Auler Junior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

REVIEW

Operative versus nonoperative treatment for displaced midshaft clavicle fractures: a meta-analysisbased on current evidenceXin-Hua Wang, Wei-Jun Guo, A-Bing Li, Guang-Jun Cheng, Tao Lei, You-Ming Zhao. . . . . . . . . . . . . . . . . . . . . . . . . 584

The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes inthe Chinese Han population: a meta-analysisEnting Ma, Huili Wang, Jing Guo, Ruirui Tian, Li Wei. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

The negative prognostic impact of bone metastasiswith a tumor massBirsen Yucel,I,* Mustafa Gurol Celasun,I Bilge Oztoprak,II Zekiye Hasbek,III Seher Bahar,I Turgut Kacan,IV

Aykut Bahceci,IV Mehmet Metin SekerIV

IDepartment of Radiation Oncology IIDepartment of Radiology IIIDepartment of Nuclear Medicine IVDepartment of Medical Oncology, Sivas,

Turkey.

OBJECTIVE: Typically, bone metastasis causes osteolytic and osteoblastic lesions resulting from the interactionsof tumor cells with osteoclasts and osteoblasts. In addition to these interactions, tumor tissues may grow insidebones and cause mass lesions. In the present study, we aimed to demonstrate the negative impact of a tumormass in a large cohort of patients with bone metastatic cancer.

METHODS: Data from 335 patients with bone metastases were retrospectively reviewed. For the analysis, allpatients were divided into three subgroups with respect to the type of bone metastasis: osteolytic, osteoblastic,or mixed. The patients were subsequently categorized as having bone metastasis with or without a tumor mass,and statistically significant differences in median survival and 2-year overall survival were observed betweenthese patients (the median survival and 2-year overall survival were respectively 3 months and 16% in patientswith a tumor mass and 11 months and 26% in patients without a tumor mass; po0.001).

RESULTS: According to multivariate analysis, the presence of bone metastasis with a tumor mass was found tobe an independent prognostic factor (p=0.011, hazard ratio: 1.62, 95% confidence interval: 1.11–1.76). Bonemetastasis with a tumor mass was more strongly associated with osteolytic lesions, other primary diseases(except for primary breast and prostate cancers), and spinal cord compression.

CONCLUSION: Bone metastasis with a tumor mass is a strong and independent negative prognostic factor forsurvival in cancer patients.

KEYWORDS: Bone metastasis; Bone metastasis with a tumor mass; Prognostic factor; Survival.

Yucel B, Celasun MG, Oztoprak B, Hasbek Z, Bahar S, Kacan T, et al. The negative prognostic impact of bone metastasis with a tumor mass.Clinics. 2015;70(8):535-540

Received for publication on February 6, 2015; First review completed on March 23, 2015; Accepted for publication on March 31, 2015

E-mail: [email protected]

*Corresponding author

’ INTRODUCTION

Bone metastasis is the most frequent complication of cancer,occurring in up to 70% of patients with breast or prostate cancerand in approximately 15–30% of patients with carcinoma of thelung, colon, stomach, bladder, rectum, thyroid, or kidney (1).Although the exact incidence of bone metastasis remainsunknown, this type of metastasis is an attractive area of studygiven its high prevalence in cancer patients.Bone metastases develop as a result of interactions between

tumor cells and bone cells. Cancer cells can induce variousmetastatic bone lesions through different mechanisms thatdepend on the primary disease, and two types of metastatic

bone lesions have been described (2,3). The first is an osteolyticlesion that progresses with bone resorption as a result ofosteoclast activation; the second is an osteoblastic lesion thattriggers bone formation and osteoblastic cell activation. Thesetwo types of lesions may be present concomitantly in certainpatients (mixed type) following stimulation of the two differenttypes of bone cells. Alternatively, the tumor itself may growinside the bone tissue and destroy the bone directly (4). Thesemass lesions may cause an increase in complications (e.g., spinalcord compression, pathologic fracture) due to metastasis-relatedbone destruction and suggest the presence of a significant tumorburden. Examples of computerized tomography images ofosteolytic lesions, osteoblastic lesions, and bone metastasis witha tumor mass are shown in Figure 1.Although the duration of survival varies according to the

primary tumor, bone metastases are usually incurable (5).General treatment procedures for patients with bonemetastasis include bisphosphonate administration, che-motherapy, and palliative radiation therapy. However,responses to these treatment modalities are relatively poor,and the patient’s quality of life is generally impaired.DOI: 10.6061/clinics/2015(08)01

Copyright & 2015 CLINICS – This is an Open Access article distributed under theterms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in anymedium or format, provided the original work is properly cited.

No potential conflict of interest was reported.

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CLINICAL SCIENCE

Prognosis may vary among patients depending on factorssuch as the primary disease type, age, the patient’sperformance status, the metastatic interval, and the numberof metastatic sites (6,7). Nevertheless, these factors are notparticularly helpful with respect to decision making inroutine clinical practice. Moreover, data on both theprognostic impact of the mechanism type on bone metastasisand the additional role of tumor masses in these patients arelacking.Therefore, we designed a retrospective analysis to evaluate

the impact of bone metastasis-related tumor mass on patientsurvival. We also evaluated differences in the response toradiation therapy, in complications, and in the pain responsein our cohort according to the type of metastasis.

’ MATERIALS AND METHODS

This study was conducted at the Department of RadiationOncology at Cumhuriyet University Hospital in Sivas,Turkey, in accordance with the principles of the Declarationof Helsinki. A total of 335 cancer patients with bonemetastasis who were admitted to the department between2007 and 2013 were evaluated retrospectively.All patients were treated with palliative radiotherapy and

bisphosphonate. During the treatment period, all patientswere examined by a radiation oncologist immediately beforeand 1 month after radiotherapy. The physical examinationfindings as well as body weight; Eastern CooperativeOncology Group (ECOG) performance scores; and histo-pathological, radiological, and laboratory data (alkalinephosphatase [ALP] and calcium levels) were recorded. Thepatients’ survival data were obtained from hospital records,and patients lost to follow-up were contacted to obtaininformation about their condition. Survival was defined asthe time between the date of the first detection of bonemetastasis and the date of last contact or death.The cancer type was classified based on the primary site:

head and neck, lung, breast, prostate, gastrointestinalsystem, genitourinary system, or other. Prior to palliativeradiotherapy, each patient’s performance status was scoredaccording to the ECOG scoring system (8). Weight loss wasdefined as loss of 410% of body weight in 1 month.Bone metastasis was revealed by computerized tomogra-

phy or magnetic resonance imaging and was confirmed bybone scintigraphy and positron emission tomography.All patients were divided into three subgroups with

respect to the type of bone metastasis: osteolytic, osteo-blastic, or mixed type. All patients were subsequently

recategorized into two groups: bone metastasis with orwithout a tumor mass.

Pain intensity was evaluated using visual analog scales in139 (41%) of the cases (9). Patients were routinely asked torate their pain intensity by placing a mark on a 10-mm visualanalog scale at the start of radiotherapy and at 1 month afterradiotherapy. This scaling system was used to evaluate theintensity of pain only in the radiotherapy-affected region.The response to radiotherapy was determined by calculatingthe difference between the pain intensity on the visual analogscale before and 1 month after the initiation of radiotherapy.

Statistical Package for Social Sciences (SPSS) for Windows14.0 (SPSS, Inc., Chicago, IL, USA) was used for the statisticalanalysis. For descriptive statistics, the mean, standarddeviation, frequency, and median were used. Categoricaldata were compared statistically using the chi-square test orFisher’s exact test. Survival rates were calculated accordingto the Kaplan-Meier method. A multivariate analysis (Coxregression analysis) was used to evaluate independent riskfactors affecting survival. P-values p0.05 were accepted asstatistically significant.

’ RESULTS

The study group comprised 234 (70%) men and 101 (30%)women. The median age at the time of cancer diagnosis was59 years (range, 21–82 years). The primary disease distribu-tion was as follows: lung cancer in 107 (32%) patients, breastcancer in 64 (19%), prostate cancer in 62 (19%), gastro-intestinal system tumors in 40 (12%), genitourinary systemtumors in 20 (6%), head and neck tumors in 11 (3%), andtumors in other organs in 31 (9%).

Osteolytic bone metastasis was observed in 99 (30%)patients, whereas 155 (46%) had osteoblastic bone metastasis,and 71 (21%) had mixed-type bone metastasis. Ten (3%)patients had bone metastasis and only a tumor mass,without any other lesions; these 10 patients were excludedwhen categorizing the patients with respect to the type ofbone lesion (i.e., osteolytic, osteoblastic, or mixed). Bonemetastasis with a tumor mass was present in 73 (22%) cases.Eleven (3%) patients had a single bone metastatic lesion, and324 (97%) had two or more lesions. The 11 patients withsingle bone lesions had no metastases in other organs. Thelocations and frequencies of bone metastases were as follows:vertebral column metastasis in 283 (84%) patients, pelvicbone metastasis in 246 (73%), long bone metastasis in 189(56%), costal metastasis in 189 (56%), and skull metastasis in63 (19%).

Figure 1 - Types of bone metastasis (white arrows). A) Osteolytic metastasis. B) Osteoblastic metastasis. C) Bone metastasis with a tumormass.

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Spinal cord compression was observed in 20 patients, or7% of all patients with vertebral column metastases (N: 283),whereas 49 (15%) patients had pathologic fractures, 26 (8%)had neurological deficits, and 16 (5%) had hypercalcemia.Surgical interventions were performed for pathologic frac-tures in 19 (39%) patients with pathologic fractures (N: 49).The types of bone metastasis with respect to primary

disease were as follows. Among patients with lung cancer, 42(39%) had osteolytic lesions, 44 (41%) had osteoblasticlesions, 19 (18%) had mixed lesions, and 2 (2%) had bonemetastases with only tumor masses. For patients with breastcancer, 22 (34%), 19 (30%), and 22 (34%) had osteolytic,osteoblastic, and mixed lesions, respectively; 1 (2%) had abone metastasis with only a tumor mass. Osteolytic,osteoblastic, and mixed lesions developed in 2 (3%), 53(86%), and 7 (11%), respectively, patients with prostatecancer. Regarding patients with gastrointestinal systemtumors, 9 (22%), 18 (45%), and 11 (28%) had osteolytic,osteoblastic, and mixed lesions, respectively, and 2 (5%)showed bone metastases with only tumor masses. Amongpatients with genitourinary system tumors, 8 (40%), 5 (25%),and 5 (25%) had osteolytic, osteoblastic, and mixed lesions,respectively, with 2 (10%) exhibiting bone metastases withonly tumor masses. The incidence of osteolytic, osteoblastic,and mixed lesions was 2 (18%), 5 (46%), and 2 (18%),respectively, for the patients with head and neck tumors; 2(18%) had bone metastases with only tumor masses.

Bone metastasis with a tumor mass was observed morefrequently in patients with osteolytic lesions than in thosewith other bone lesions. Spinal cord compression wasobserved more frequently in cases of bone metastasis witha tumor mass compared to cases without a tumor mass;when occurring in the latter, the compression was mostlydue to compression fracture, as observed for osteolyticmetastases, or to new bone formation, as observed inosteoblastic lesions. However, serum ALP levels were higherin patients without tumor masses. In addition, bonemetastases with tumor masses were observed less frequentlyin patients with primary breast or prostate cancer comparedwith patients with other primary diseases, such as lung orgastrointestinal system tumors. With respect to pathologicfractures, pain severity, and responses to radiotherapy, nodifferences were observed between cases of bone metastaseswith tumor masses and cases of other bone metastases(Table 1).The median survival duration was 10 months (range, 1–

147 months), and the 1- and 2-year survival rates were 46%and 24%, respectively. The median survival duration was 3months and the 1- and 2-year survival rates were 28% and16%, respectively, among patients who had bone metastaseswith tumor masses and 11 months and 50% and 26%,respectively, in patients who had bone metastasis withouttumor masses. The survival curves of the patients with orwithout a tumor mass are shown in Figure 2. Univariate

Table 1 - Comparison of features associated with bone metastases with or without tumor masses.

Bone metastasis without a tumor mass(N: 262, 78%)

Bone metastasis with a tumor mass(N: 73, 22%)

p-value

Type of bone metastasisOsteolytic 60 (61) 39 (39) o0.001Osteoblastic 151 (97) 4 (3)Mixed 51 (72) 20 (28)

Bone metastasis with only a tumor mass - 10 (100)Primary diseaseLung 80 (75) 27 (25) o0.001Breast 57 (89) 7 (11)Prostate 58 (94) 4 (6)Gastrointestinal system 27 (68) 13 (32)Genitourinary system 13 (65) 7 (35)Head and neck 7 (64) 4 (36)Other 20 (65) 11 (35)

Serum ALP1 levelp129 U/L 137 (75) 47 (25) 0.0284129 U/L 119 (84) 23 (16)

Serum calcium levelp10.6 mg/dL 246 (79) 64 (21) 0.103410.6 mg/dL 10 (63) 6 (37)

Spinal cord compressionNo 219 (83) 44 (17) o0.001Yes 6 (30) 14 (70)

Pathologic fractureNo 228 (80) 58 (20) 0.079Yes 34 (69) 15 (31)

SurgeryNo 249 (79) 67 (21) 0.213Yes 13 (68) 6 (32)

Severity of painMild 12 (86) 2 (14) 0.312Moderate 25 (66) 13 (34)Severe 57 (65) 30 (35)

Response to radiotherapyNo 23 (64) 13 (36) 0.306Yes 71 (69) 32 (31)

Abbreviation: 1ALP, alkaline phosphatase

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analyses showed that the survival duration after metastasiswas affected by the presence of bone metastasis with a tumormass as well as by gender, weight loss, performance status,serum ALP and calcium levels, primary disease, bonemetastasis type, number of bone lesions, the presence ofextraosseous metastasis, and the disease-free interval. Theprognostic factors that affected survival time after thedevelopment of bone metastasis are shown in Table 2.Multivariate analyses revealed that the presence of bone

metastasis with a tumor mass as well as gender, weight loss,primary disease, type of bone metastasis, and serum ALPand calcium levels were independent prognostic factors thataffected survival. The independent prognostic factors thataffected the duration of survival after the development ofbone metastasis are shown in Table 3.

’ DISCUSSION

The prevalence of bone metastasis is higher in advanced-stage cancers. Patients diagnosed with bone metastasisusually have incurable disease, though the survival durationdoes vary based on the primary disease. Accordingly, it isvery important to determine prognostic factors once adiagnosis of bone metastasis has been made. The presentstudy investigated the prognostic and clinical importance ofbone metastasis with a tumor mass and found that thisfeature was an apparently strong negative prognostic factorfor survival. The higher incidence of these metastases inassociation with osteolytic lesions might have contributed tothis result, as the presence of osteolytic lesions was found tobe a poor prognostic factor in a multivariate analysis. Inaddition, growth of the tumor itself inside the bone mightindicate a larger tumor burden, which might also contributeto a shorter survival duration. Given the soft tissuecomponent of bone metastasis with a tumor mass, spinalcord compression was observed more frequently in thesepatients; nonetheless, the presence of these lesions did not

increase the pain intensity or affect the response toradiotherapy.

Certain researchers have studied prognostic factors inpatients with bone metastases. In a study of 350 patients withskeletal metastases, Katagiri et al. (6) reported that thepatient’s performance status, the primary lesion site, thepresence of multiple skeletal metastases, the presence ofvisceral or cerebral metastases, and a history of previouschemotherapy were important prognostic factors. Van derLinder et al. (7) reported a median survival time of 7 monthsfor 342 patients with vertebral metastases, and Karnofskystated that the performance score, the primary tumor type,and absence of visceral metastasis were significant predictorsof survival. In the present study, female gender, the presenceof osteoblastic and/or mixed lesions, and primary breast orprostate cancer were considered to be good prognosticpredictors. In contrast, the presence of bone metastasis with atumor mass as well as male gender, weight loss, primarylung cancer, the presence of osteolytic lesions, and elevatedALP and calcium levels were found to be poor prognosticpredictors. Poor performance in a single-variable analysis, adisease-free interval of o2 years, the presence of extraoss-eous metastasis, and multiple bone lesions were also poorprognostic factors.

Circulating metastatic cells in blood become entrapped bythe bone marrow spongiosum. Cancerous bone undergoessecondary lytic or blastic changes (10), and the type of bonemetastasis is determined by these changes. In the literature,osteolytic lesions have been reported to be more frequent inbreast cancer cases, whereas osteoblastic lesions are observedin cases of prostate cancer. In the present study, osteoblasticlesions (46%) were more frequently observed in the overallpatient population; similar to the findings of other studies,osteolytic lesions were more frequent in patients with breastcancer, with osteoblastic lesions being more common inpatients with prostate cancer. In terms of the conventionalclassification of bone metastases, the presence of a tumor

Months140120100806040200

100

80

60

40

20

0

Yes-censoredYes-censore

No-censoredNo-

YesYe

NoNo

Tumor mass

Survival Functions

Cum

surv

ival

Figure 2 - Survival curves of patients with or without a tumor mass.

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mass was significantly more frequent among osteolyticlesions (62%). The frequencies of bone metastasis with atumor mass were low among patients with breast or prostatecancer and similar among those with other types of cancer.Specifically, 25–36% of patients with other types of cancer(non-breast or prostatic) had bone lesions with tumormasses.Bone metastases are associated with a particular set of

complications, and the frequency of these complicationsvaries depending on the features of the metastatic lesions.For example, pathologic fractures and spinal cord compres-sion are encountered more frequently with osteolytic lesions,as these lesions cause bone destruction (2,11). It is rational toexpect that bone metastases with tumor masses wouldpresent more complications; indeed, spinal cord compressionwas more frequent among cases of bone metastasis with atumor mass in the current study. However, an elevatedserum ALP level was more frequently observed in cases ofbone metastasis without a tumor mass. In terms ofpathologic fractures, serum calcium levels, surgical interven-tion, pain severity, and responses to radiotherapy, nodifferences were observed between patients with bonemetastasis with a tumor mass and those with other typesof bone metastases.

The survival duration in patients with bone metastasesvaried quite significantly depending on the primary disease,and it is reported that the duration is generally longer forpatients with breast or prostate cancer than for those withother types of cancer (1,7,6,11). Ahn et al. (12) reported amedian survival time of 55.2 months among 110 breastcancer patients with only bone metastases. In contrast,survival durations as short as 5–7 months were reportedamong patients with lung cancer and bone metastases(11,13,14). In our study, the longest survival durations wereobserved in patients with breast cancer, followed by thosewith prostate cancer (median survival durations of 18months and 15 months, respectively); conversely, thesurvival times of patients with other cancers were relativelyshort.Many studies have reported that patients with single bone

lesions in the absence of metastases in other organs have alonger survival duration relative to those with multiple bonemetastases (15-17). In a study of 42 patients with solitarybone metastases, Hoshi et al. (15) reported a median survivalduration of 30 months and a 1-year survival rate of 76.5%. Inthe present study, the 11 patients with single bone lesionshad a median survival duration of 32 months and a 1-yearsurvival rate of 68%. The survival durations were shorter

Table 2 - Prognostic factors affecting patient survival after the development of bone metastasis, as determined by univariate survivalanalysis.

No. of patients 1-year survival (%) 2-year survival (%) Median survival (months) p-value

Bone metastasis with tumor massNo 262 50 26 11 *o0.001Yes 73 28 16 3

GenderMale 234 39 17 8 o0.001Female 101 61 42 17

Weight lossNo 248 53 27 12 o0.001Yes 87 24 12 5

ECOG PS1

ECOG0-1 168 55 30 13 o0.001ECOG2 and higher 167 36 17 7

Serum ALP2 levelp129 U/L 184 50 29 12 0.0044129 U/L 142 39 16 9

Serum calcium levelp10.6 mg/dL 310 46 24 10 0.027410.6 mg/dL 16 - - 3

Primary diseaseLung 107 27 10 5 o0.001Breast 64 72 47 18Prostate 62 69 31 15Gastrointestinal system 40 24 6 5Genitourinary system 20 20 10 5Head and neck 11 9 - 3

Type of bone metastasisOsteolytic 99 29 14 4 0.004Osteoblastic 155 53 26 12Mixed 71 49 26 12

Number of bone lesions1 lesion 11 68 68 32 0.040X2 lesions 324 44 22 10

Extraosseous metastasisNo 176 51 27 12 0.032Yes 159 40 18 8

Disease-free intervalo24 months 259 41 20 9 0.026X24 months 76 61 35 18

Abbreviations: 1ECOG PS, Eastern Cooperative Oncology Group performance status; 2ALP, alkaline phosphatase

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among the patients with osteolytic lesions compared withpatients with osteoblastic or mixed lesions. Moreover,patients with bone metastases with tumor masses hadsignificantly shorter survival durations compared with thosewith bone metastases without tumor masses (mediansurvival durations of 3 months and 11 months, respectively;1-year survival rates of 28% and 50%, respectively).Two major limitations of the present study were its

retrospective design and its heterogeneous study population.We believe that studies of more specific groups would yieldmore significant results.The presence of bone metastasis with a tumor mass

appeared to be a strong negative prognostic factor and wasassociated with a higher incidence of spinal cord compression.

’ AUTHOR CONTRIBUTIONS

Yücel B designed the research and analyzed the data. Yücel B, CelasunMG, Öztoprak B, Hasbek Z, Bahar S, Kacan T, Bahceci A, and Seker MMperformed the research. Kacan T and Seker MM contributed analyticaltools. Yücel B and Öztoprak B wrote the paper. The authors have nofinancial disclosures to declare, no conflicts of interest to report, and haveno commercial or proprietary interest.

’ REFERENCES

1. Coleman RE, Rubens RD. The clinical course of bone metastases frombreast cancer. Br J Cancer. 1987;55(1):61-6, http://dx.doi.org/10.1038/bjc.1987.13

2. Roodman GD. Mechanisms of bone metastasis. N Engl J Med. 2004;350(16):1655-64, http://dx.doi.org/10.1056/NEJMra030831

3. Mundy GR. Metastasis to bone: causes, consequences and therapeuticopportunities. Nat Rev Cancer. 2002;2(8):584-93, http://dx.doi.org/10.1038/nrc867

4. Käkönen SM, Mundy GR. Mechanisms of osteolytic bone metastases inbreast carcinoma. Cancer. 2003;97(3Suppl):834-9, http://dx.doi.org/10.1002/(ISSN)1097-0142

5. Coleman RE. Metastatic bone disease: clinical features, pathophysiologyand treatment strategies. Cancer Treat Rev.2001;27(3):165-76, http://dx.doi.org/10.1053/ctrv.2000.0210

6. Katagiri H, Takahashi M, Wakai K, Sugiura H, Kataoka T, Nakanishi K.Prognostic factors and a scoring system for patients with skeletal metas-tasis. J Bone Joint Surg Br. 2005;87(5):698-703, http://dx.doi.org/10.1302/0301-620X.87B5.15185

7. Van der Linder YM, Dijkstra SPDS, Vonk EJA, Marijnen CAM, Leer JWH.Prediction of survival in patients with metastases in the spinal column.Cancer.2005;103(2):320-8, http://dx.doi.org/10.1002/(ISSN)1097-0142

8. Oken MM, Creech RH, Tormey DC, Horton J, Davis T, Mc Fadden ET,et al. Toxicity and response criteria of the Eastern Cooperative OncologyGroup. Am J ClinOncol. 1982:5(6):649-55, http://dx.doi.org/10.1097/00000421-198212000-00014

9. Price DD, McGrath PA, Rafii A, Buckingham B. The validation of visualanalogue scales as ratio scale measures for chronic and experimental pain.Pain. 1981;17(1):45-56, http://dx.doi.org/10.1016/0304-3959(83)90126-4

10. Rubin P, Brasacchio R, Katz A. Solitary metastases: illusion versus reality.Semin Radiat Oncol. 2006;16(2):120-30, http://dx.doi.org/10.1016/j.semradonc.2005.12.007

11. Coleman RE. Clinical features of metastatic bone disease and risk ofskeletal morbidity. Clin Cancer Res. 2006;66(12):6243-9, http://dx.doi.org/10.1158/1078-0432.CCR-06-0931

12. Ahn SG, Lee HM, Cho SH, Lee SA, Hwang SH, Jeong J, et al. Prognosticfactors for patients with bone-only metastasis in breast cancer. Yonsei MedJ. 2013;54(5):1168-77, http://dx.doi.org/10.3349/ymj.2013.54.5.1168

13. Stanley KE. Prognostic factors for survival in patients with inoperablelung cancer. J Natl Cancer Inst. 1980;65(1):25-32.

14. Sugiura H, Yamada K, Sugiura T, Hida T, Mitsudomi T. Predictors of sur-vival in patients with bone metastasis of lung cancer. Clin Orthop RelatRes. 2008;466(3):729-36, http://dx.doi.org/10.1007/s11999-007-0051-0

15. Hoshi M, Takada J, Leguchi M, Takahashi S, Nakamura H. Prognosticfactors for patients with solitary bone metastasis. Int J Clin Oncol. 2013;18(1):164-9, http://dx.doi.org/10.1007/s10147-011-0359-3

16. Koizumi M, Yoshimoto M, Kasumi M, Ogata E. Comparison between soli-tary and multiple skeletal metastatic lesions of breast cancer patients. AnnOncol. 2003;14(8):1234-40, http://dx.doi.org/10.1093/annonc/mdg348

17. Hirano Y, Oda M, Tsunezuka Y, Ishikawa N, Watanabe G. Long-termsurvival cases of lung cancer presented as solitary bone metastasis. AnnThorac Cardiovasc Surg. 2005;11(6):401-4.

Table 3 - Independent prognostic factors affecting the duration of survival after the development of bone metastasis, as determinedby multivariate analysis.

Overall survival

HR1 95% CI2 p-value

Bone metastasis with a tumor massNo 1Yes 1.62 1.11–1.76 *0.011

GenderMale 1Female 0.45 0.31–0.64 o0.001

Weight lossNo 1Yes 1.39 1.02–1.90 0.034

Primary diseaseLung 1Breast 0.32 0.20–0.57 o0.001Lung 1Prostate 0.45 0.30–0.67 0.001

Type of bone metastasisOsteolytic 1Osteoblastic 0.56 0.39–0.81 0.002Osteolytic 1Mixed 0.56 0.38–0.83 0.004

Serum ALP3 levelp129 U/L 14129 U/L 1.34 1.03–2.00 0.030

Serum calcium levelp10.6 mg/dL 1410.6 mg/dL 2.22 1.03–4.81 0.042

Abbreviations: 1HR, hazard ratio; 2CI, confidence interval; 3ALP, alkaline phosphatase

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http://dx.doi.org/10.1038/bjc.1987.13

http://dx.doi.org/10.1038/bjc.1987.13

http://dx.doi.org/10.1056/NEJMra030831

http://dx.doi.org/10.1038/nrc867

http://dx.doi.org/10.1038/nrc867

http://dx.doi.org/10.1002/(ISSN)1097-0142


http://dx.doi.org/10.1053/ctrv.2000.0210

http://dx.doi.org/10.1053/ctrv.2000.0210

http://dx.doi.org/10.1302/0301-620X.87B5.15185

http://dx.doi.org/10.1302/0301-620X.87B5.15185


http://dx.doi.org/10.1097/00000421-198212000-00014

http://dx.doi.org/10.1097/00000421-198212000-00014

http://dx.doi.org/10.1016/0304-3959(83)90126-4

http://dx.doi.org/10.1016/j.semradonc.2005.12.007

http://dx.doi.org/10.1016/j.semradonc.2005.12.007

http://dx.doi.org/10.1158/1078-0432.CCR-06-0931

http://dx.doi.org/10.1158/1078-0432.CCR-06-0931

http://dx.doi.org/10.3349/ymj.2013.54.5.1168

http://dx.doi.org/10.1007/s11999-007-0051-0

http://dx.doi.org/10.1007/s10147-011-0359-3

http://dx.doi.org/10.1093/annonc/mdg348

Ocular risk management in patients undergoing generalanesthesia: an analysis of 39,431 surgeriesNewton Kara-Junior,I,II Rodrigo Franca de Espindola,II ,* Joao Valverde Filho,I Christiane Pellegrino Rosa,I

Andre Ottoboni,I Enis Donizete SilvaI

I Sırio-Libanes Hospital, Sao Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Sao Paulo, Ophthalmology Department, Sao Paulo/SP, Brazil.

OBJECTIVE: This study sought to describe and analyze ocular findings associated with nonocular surgery inpatients who underwent general anesthesia.

METHODS: The authors retrospectively collected a series of 39,431 surgeries using standardized data forms.

RESULTS: Ocular findings were reported in 9 cases (2.3:10,000), which involved patients with a mean age of 58.9±19.5 years. These cases involved patients classified as ASA I (33%), ASA II (55%) or ASA III (11%). Generalanesthesia with propofol and remifentanil was used in 4 cases, balanced general anesthesia was used in 4 cases,and regional block was used in combination with balanced general anesthesia in one case. Five patients (55%)underwent surgery in the supine position, one patient (11%) underwent surgery in the lithotomy position, twopatients (22%) underwent surgery in the prone position, and one patient (11%) underwent surgery in thelateral position. Ocular hyperemia was detected in most (77%) of the 9 cases with ocular findings; pain/burningof the eyes, visual impairment, eye discharge and photophobia were observed in 55%, 11%, 11% and 11%,respectively, of these 9 cases. No cases involved permanent ocular injury or vision loss.

CONCLUSION: Ophthalmological findings after surgeries were uncommon, and most of the included patientswere relatively healthy. Minor complications, such as dehydration or superficial ocular trauma, should beprevented by following systematic protocols that provide appropriate ocular occlusion with a lubricatingointment and protect the eye with an acrylic occluder. These procedures will refine the quality of anesthesiaservices and avoid discomfort among patients, surgeons and anesthesia staff.

KEYWORDS: Blindness; Anesthesia; Eye Injuries.

Kara-Junior N, Espindola RF, Valverde Filho J, Rosa CP, Andre Ottoboni, Silva ED. Ocular risk management in patients undergoing generalanesthesia: an analysis of 39,431 surgeries. Clinics. 2015;70(8):541-543

Received for publication on January 22, 2015; First review completed on April 7, 2015; Accepted for publication on May 12, 2015



’ INTRODUCTION

Postoperative visual loss (POVL) following general sur-gery is a relatively uncommon but devastating complicationthat is most frequently associated with cardiac, spine, headand neck operations. Estimates have indicated that POVLoccurs in up to 0.2% (1) and 4.5% (2) of spine and cardiacsurgeries, respectively.Although studies of 65,000 and 400,000 patients who

underwent anesthesia for all types of surgery at two largeacademic institutions suggested a low prevalence of perio-perative vision loss in surgeries other than cardiac and spinalfusion procedures, the actual prevalences of perioperativevision loss for the most common types of operations remainunknown (3,4).

Because the frequency of ocular complications is very low,few peer-reviewed studies have analyzed ocular symptomsand vision loss after surgeries under general anesthesia. Theaim of this retrospective study was to contribute to theprevention of ocular complications during anesthesia bydetermining and analyzing the ocular findings from a largeseries of cases involving general anesthesia.


This retrospective study included 39,431 nonocular sur-geries. We began the study by reviewing the documentedcases of ocular findings after surgical procedures performedat our institution between January 2007 and December 2010.The preoperative variables included age, sex, American

Society of Anesthesiologists (ASA) physical status classifica-tion, urgency of surgery (emergency or elective), duration ofthe procedure, ocular findings (signs and symptoms) andsurgical position during surgery. Other variables includedthe use of ocular lubricant during anesthesia, the requiredtreatment and the final diagnosis.DOI: 10.6061/clinics/2015(08)02


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All demographic variables were analyzed using descrip-tive statistics; in particular, means and SDs were determinedfor continuous variables, and frequencies (in percentages)were calculated for categorical variables.

’ RESULTS

This retrospective study included 39,431 nonocular surgeries.Ocular findings were reported in 9 cases (2.3:10,000), whichinvolved patients with a mean age of 58.9±19.5 years. Table 1presents the characteristics of all 9 cases. Examinations ofindividual variables revealed that male patients (66%), ASA IIstatus (55%), elective surgery (88%) and the supine position(66%) were each involved in the majority of these cases.For patients with ocular findings who were subjected to

general anesthesia (9 cases), pain (55%) and photophobia(22%) were the main symptoms, and hyperemia (77%) wasthe main sign (Table 2). Only one patient presented withblurred vision (11%).The main diagnoses in these cases were direct trauma

(44%) and dry eye (33%) (Table 2). All 9 patients experiencedocular occlusion during surgery, and 5 patients (55%) alsoreceived lubricant. No patient exhibited permanent ocularinjury or significant visual loss.

’ DISCUSSION

Perioperative ischemic optic neuropathy (PION) has beenreported after spine (5-7), orthopedic (8), neck (9-13), heart,and abdominal surgeries (14,15). Intraoperative variablesthat reportedly play roles in the pathogenesis of PIONinclude hypotension, anemia, and elevated intraocularpressure associated with the prone position during spinalsurgery (16). Vascular risk factors, such as diabetes, coronaryartery disease, and hypertension, are present in manypatients who experience PION (17,18), although vision losshas been reported in children and healthy adults who exhibitnone of these factors (6).Given that the mechanisms and risk factors for PION are

poorly understood, the risks of vision loss should be consideredin preoperative discussions with patients who expect toundergo spine surgery or surgery requiring cardiopulmonarybypass because such procedures are associated with the highestincidences of this rare complication (19).In the present study, the incidence of ocular findings was

2.3:10.000. No patient experienced permanent ocular injuryor significant visual loss. However, certain of the observedsymptoms/signs could significantly impact eye health.All 9 of the patients with ocular findings experienced

ocular occlusion during their procedures, and 55% of these

patients received ocular lubricant (in the form of eye drops,serum or gel). These findings indicated the precautionsimplemented by the staff to prevent ocular injury. However,these actions cannot avoid ocular complications in all cases.

The most common diagnoses found in our study weredirect trauma and dry eye. Preventive strategies are the onlyoption to reduce the effect of ocular complications duringgeneral anesthesia. Ocular occlusion and the use of eye-lubricating ointment are important strategies to preventdehydration of the ocular surface during long surgeries. Inthese situations, the mechanisms of aggravation can includenot only corneal exposure if the eyelids remain open but alsodecreased tear secretion induced by the anesthesia. After thesurgical procedure, the maintenance of the patient’s ocularocclusion is recommended during the postanesthetic period,when the blink reflex is poor and the patient remains sleepy.The main symptoms of dry eye are pain, redness and tearing.With respect to the treatment of corneal deepithelializationdue to exposure keratopathy, eye lubrication with artificialtears and occlusion with ocular lubricating ointment arefrequently recommended in severe cases. The ocular admin-istration of saline should be avoided due to the risk of furtherdehydration of the cornea. Frequent review of dry eye casesby an ophthalmologist is necessary due to the risk ofprogression to ulceration of the cornea, which can lead topermanent vision loss.

Direct trauma to the eye is generally caused by pressureexerted by the surgeon’s arm or hand on the patient’s eyeduring surgery or by direct corneal injury with instrumentsor components of the surgical field. During intubation, theanesthetist himself may cause trauma to the patient’s eye.Ocular trauma can be prevented by the systematic use ofacrylic eye protection similar to the postoperative protectionused after eye surgery to prevent patients from exertingpressure on their eyes; the use of such protection may beparticularly important for patients whose surgical sites arenear the head (20).

In the present study, there were no cases of permanentvision loss. Many strategies can be used to prevent blindness,particularly PION-related blindness. Maneuvers to keep thehead at or above heart level to reduce venous congestion inthe head have been recommended in the ASA practiceadvisory for perioperative visual loss associated with spinesurgery (21). Minimizing the duration of time in the proneposition and maximizing hemostasis may also be beneficial.

In summary, an understanding of the risk factors andcharacteristics that promote the occurrence of perioperativeocular lesions is extremely important for the development ofprevention strategies. Despite the low incidence of thesecomplications, the potential for serious and permanent visual

Table 1 - Patient characteristics.

Patient Sex Age Duration (min) ASA Surgery Anesthesia Position

1 male 69 105 I elective balanced general anesthesia supine2 male 21 450 I elective general anesthesia – propofol and remifentanil supine3 female 69 345 I elective balanced general anesthesia supine4 male 77 150 II elective general anesthesia – propofol and remifentanil prone5 female 34 255 II emergency general anesthesia – propofol and remifentanil supine6 male 62 275 II elective general anesthesia – propofol and remifentanil lithotomy7 female 60 345 II elective balanced general anesthesia prone8 male 58 135 II elective balanced general anesthesia supine9 male 80 135 III elective general anesthesia – propofol and remifentanil supine

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injuries such as retinal ischemia and PION justify appro-priate care and the active pursuit of high-quality anesthesiaservices. Since 2010, a protocol involving ocular occlusionwith the instillation of lubricant eye drops during relativelycomplex procedures has been systematically adopted by theanesthesia services of Sírio Libanês Hospital. Beginning in2014, guided by the results and insights of this study, whichwas conducted and analyzed in collaboration with ophthal-mologists, lubricating ointment and ocular occlusion with anacrylic occluder for eye protection have been used for allsurgeries involving general anesthesia. It is recommendedthat these procedures, which have been implemented toachieve the objective of further improving patient safetyduring surgery, should be followed from the induction ofanesthesia to the complete awakening of the patient in thepostanesthesia recovery room.Minor complications, such as dehydration or superficial

ocular trauma, which can generally be rapidly resolvedduring the postoperative period, should be prevented byfollowing systematic protocols that include appropriateocular occlusion with lubricating ointment and protectionof the eye with an acrylic occluder. These protocols willrefine the quality of anesthesia services and avoid discomfortamong patients, surgeons and anesthesia staff.


Kara-Junior N: study conception and design; drafting of the manuscript;and critical revision. Espindola RF: drafting of the manuscript; criticalrevision; and analysis and interpretation of study data. Valverde Filho J,Rosa CP, Ottoboni A, and Silva ED: study conception and design; dataacquisition; and analysis and interpretation of study data.

’ REFERENCES

1. Stevens WR, Glazer PA, Kelley SD, Lietman TM, Bradford DS. Ophthal-mic complications after spinal surgery. Spine. 1997;22(12):1319–24.

2. Shaw PJ, Bates D, Cartlidge NE, Heaviside D, French JM, Julian DG, et al.Neuro-ophthalmological complications of coronary artery bypass graftsurgery. Acta Neurol Scand. 1987;76(1):1–7.

3. Roth S, Thisted RA, Erickson JP, Black S, Schreider BD. Eye injuries afternon ocular surgery: a study of 60,965 anesthetics from 1988 to 1992.Anesthesiology 1996;85(5):1020–7.

4. Warner ME, Warner MA, Garrity JA, MacKenzie RA, Warner DO. Thefrequency of perioperative vision loss. Anesth Analg. 2001;93(6):1417–21.

5. Katz DM, Trobe JD, Cornblath WT, Kline LB: Ischemic optic neuropathyafter lumbar spine surgery. Arch Ophthalmol. 1994;112(7):925–31.

6. Alexandrakis G, Lam BL. Bilateral posterior ischemic optic neuropathyafter spinal surgery. Am J Ophthalmol. 1999;127(3):354–5.

7. Cheng MA, Sigurdson W, Tempelhoff R, Lauryssen C. Visual loss afterspine surgery: A survey Neurosurgery. 2000;46(3):625–31.

8. Roth S, Nunez R, Schreider BD. Unexplained visual loss after lumbarspinal fusion. J Neurosurg Anesthesiol. 1997;9(4):346–8.

9. Bhatti MT, Enneking FK. Visual loss and ophthalmoplegia after shouldersurgery. Anesth Analg. 2003;96(3):899–902.

10. Marks SC, Jaques DA, Hirata RM, Saunders JR Jr. Blindness followingbilateral radical neck dissection. Head Neck. 1990;12(4):342–5.

11. Nawa Y, Jaques JD, Miller NR, Palermo RA, Green WR. Bilateral posterioroptic neuropathy after bilateral radical neck dissection and hypotension.Graefes Arch Clin Exp Ophthalmol. 1992;230(4):301–8.

12. Schobel GA, Schmidbauer M, Millesi W, Undt G. Posterior ischemic opticneuropathy following bilateral radical neck dissection. Int J OralMaxillofac Surg. 1995;24(4):283–7.

13. Worrell L, Rowe M, Petti G. Amaurosis: A complication of bilateral radicalneck dissection. Am J Otolaryngol. 2002;23(1):56–9.

14. Pazos GA, Leonard DW, Blice J, Thompson DH. Blindness after bilateralneck dissection: Case report and review. Am J Otolaryngol. 1999;20(5):340–5.

15. Asensio JA, Forno W, Castillo GA, Gambaro E, Petrone P. Posteriorischemic optic neuropathy related to profound shock after penetratingthoracoabdominal trauma. South Med J. 2002;95(9):1053–7.

16. Johnson MW, Kincaid MC, Trobe JD. Bilateral retrobulbar optic nerveinfarctions after blood loss and hypotension. A clinicopathologic casestudy. Ophthalmology. 1987; 94(12):1577–84.

17. Cheng MA, Todorov A, Tempelhoff R, McHugh T, Crowder CM,Lauryssen C. The effect of prone positioning on intraocular pressure inanesthetized patients. Anesthesiology. 2001;95(6):1351–5.

18. Kim JW, Hills WL, Rizzo JF, Egan RA, Lessell S. Ischemic optic neuro-pathy following spine surgery in a 16-year-old patient and a ten-year-oldpatient. J Neuroophthalmol. 2006;26(1):30–3.

19. Holy SE, Tsai JH, McAllister RK, Smith KH. Perioperative Ischemic OpticNeuropathy. A Case Control Analysis of 126,666 Surgical Procedures at aSingle Institution. Anesthesiology. 2009; 110(2):246–53.

20. Carvalho RS, Kara-Jose N, Temporini ER, Kara-Junior N, Noma RK. Self-medication: the first attempt in patients seen in an ophthalmologicemergency room. Clinics. 2009;64(8):735–41.

21. American Society of Anesthesiologists Task Force on PerioperativeBlindness: Practice advisory for perioperative visual loss associated withspine surgery: A report by the American Society of Anesthesiologists TaskForce on Perioperative Blindness. Anesthesiology. 2006;104(6):1319–28.

Table 2 - Description of ocular findings (signs and symptoms), treatments and final diagnoses for patients subjected to generalanesthesia.

Patient Use of ocularlubricant

Type oflubricant

Sign(s) Symptom(s) Treatment(s) Diagnosis

1 yes eye drops hyperemia,edema

pain antibiotics direct trauma

2 yes serumphysiologicalsolution

hyperemia pain antibiotics, mydriatic drugs,corticosteroids

direct trauma/dehydration by serum

3 no - discharge - serum physiological solution dry eye4 yes gel hyperemia pain lubrication, antibiotics corneal

deepithelialization5 no - - - cold compress, lubrication,

corticosteroidsdry eye

6 yes gel hyperemia photophobia lubrication exposure keratopathy7 no - hyperemia,

palpebral edemapain; photophobia;blurred vision

eye anesthetics, antibiotics,lubrication, cold compress

direct trauma

8 yes ointment hyperemia pain eye anesthetics, lubrication, coldcompress

direct trauma

9 no - hyperemia - corticosteroids dry eye

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CLINICS 2015;70(8):541-543 Ocular risk in general anesthesiaKara-Junior N et al.

Flow-through anastomosis using a T-shaped vascularpedicle for gracilis functioning free muscle transplanta-tion in brachial plexus injuryYi Hou, Jiantao Yang, Yi Yang, Bengang Qin, Guo Fu, Xiangming Li, Liqiang Gu*, Xiaolin Liu,

Qingtang Zhu, Jian Qi

The First Affiliated Hospital of Sun Yat-sen University, Department of Microsurgery, Orthopedic Trauma, and Hand Surgery, Guangzhou, China

OBJECTIVE: In gracilis functioning free muscle transplantation, the limited caliber of the dominant vascularpedicle increases the complexity of the anastomosis and the risk of vascular compromise. The purpose of thisstudy was to characterize the results of using a T-shaped vascular pedicle for flow-through anastomosis ingracilis functioning free muscle transplantation for brachial plexus injury.

METHODS: The outcomes of patients with brachial plexus injury who received gracilis functioning free muscletransplantation with either conventional end-to-end anastomosis or flow-through anastomosis from 2005 to 2013were retrospectively compared. In the flow-through group, the pedicle comprised a segment of the profundafemoris and the nutrient artery of the gracilis. The recipient artery was interposed by the T-shaped pedicle.

RESULTS: A total of 46 patients received flow-through anastomosis, and 25 patients received conventional end-to-end anastomosis. The surgical time was similar between the groups. The diameter of the arterial anastomosisin the flow-through group was significantly larger than that in the end-to-end group (3.87 mm vs. 2.06 mm,respectively, po0.001), and there were significantly fewer cases of vascular compromise in the flow-throughgroup (2 [4.35%] vs. 6 [24%], respectively, p=0.019). All flaps in the flow-through group survived, whereas 2 inthe end-to-end group failed. Minimal donor-site morbidity was noted in both groups.

CONCLUSIONS: Flow-through anastomosis in gracilis functioning free muscle transplantation for brachial plexusinjury can decrease the complexity of anastomosis, reduce the risk of flap loss, and allow for more variation inmuscle placement.

KEYWORDS: Brachial plexus injury; Functioning free muscle transplantation; Flow-through anastomosis;Gracilis muscle; T-shaped pedicle.

Hou Y, Yang J, Yang Y, Qin B, Fu G, Li X, et al. Flow-through anastomosis using a T-shaped vascular pedicle for gracilis functioning free muscletransplantation in brachial plexus injury. Clinics. 2015;70(8):544-549

Received for publication on February 10, 2015; First review completed on March 30, 2015; Accepted for publication on May 12, 2015



’ INTRODUCTION

Traumatic brachial plexus injury (BPI) is a severe anddevastating condition observed in up to 4.2% of multitraumavictims (1). Although transfer of multiple nerves has shownsatisfactory results for traumatic BPI (2-5), the management oftotal BPI, or brachial plexus avulsion, is challenging. Function-ing free muscle transplantation (FFMT) with or without transferof multiple nerves has been increasingly accepted as animportant option for achieving functional reconstruction (6-11).

FFMT consists of the surgical transplantation of normalmuscle with neurovascular anastomosis to replace adestroyed or denervated muscle. FFMT has been used torestore upper limb function following traumatic BPI (12),muscle loss or denervation (13), Volkmann’s ischemiccontracture (14), and tumor resection (15). In contrast tocommon free cutaneous or musculocutaneous flap transfer,FFMT aims to restore function rather than to provide simplewound coverage and soft tissue repair. In essence, FFMT is avascularized free tissue transfer, and many factors canadversely affect the outcomes of flaps, especially includingfailure of the vascular anastomosis (16). Therefore, reliableanastomosis of blood vessels is one of the most importantprerequisites for successful FFMT.

The gracilis muscle is the donor muscle most widelyused in FFMT, and the limited caliber of the vascularpedicle is problematic. In particular, the diameter of thegracilis artery is only 1.0 mm to 2.5 mm (17-19), whichDOI: 10.6061/clinics/2015(08)03



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causes difficulty in finding matched-size recipient vesselsand which also limits blood flow at the site of anastomosis.Thus, FFMT using the gracilis muscle is susceptible tovascular compromise. Moreover, BPI associated withvascular injury is not uncommon. The concomitantvascular damage, although insidious, often involves thecommonly used arterial branches in the recipient limb,leading to less-than-optimal vascular anastomosis and anincreased failure risk in FFMT. An anastomosis methodother than the conventional end-to-end or side-to-sidemethod is thus needed to decrease the risk of gracilis flapfailure in FFMT.Flow-through anastomosis uses a T-shaped vascular

pedicle to bridge the recipient blood vessels. Flaps with aflow-through design were originally used in complextrauma surgery and malignant tumor resection, whichrequire simultaneous wound coverage and blood vesselrepair. By using a T-shaped vascular pedicle, injured ordeficient vessels at the recipient site can be repaired viacirculation through the flap while the flap is simultaneouslyrevascularized by the recipient blood vessels. Foucher et al.(20) first reported a flow-through flap in 1984, and Costa etal. (21) reported 1-stage coverage and revascularization oftraumatized limbs using a flow-through flap in 1991. Sincethat time, the technique has been widely applied in traumasurgery to achieve wound coverage and vascular repair inone stage (22-24). In addition, due to its versatility, the flow-through technique has been used in various free flaptransfers to increase blood inflow, decrease overall resis-tance, and capture more variant perforators (25-30). How-ever, there are few reports of this technique’s use withFFMT.Thus, the purpose of this study was to compare the results

of flow-through anastomosis with those of traditional end-to-end anastomosis in gracilis FFMT used for the repair oftraumatic BPI.

’ METHODS

PatientsThe cases of consecutive patients with traumatic BPI

treated with gracilis FFMT at our center from 2005 to 2013were retrospectively reviewed. The criteria for inclusion inthis study were patients with traumatic BPI who receivedFFMTusing the gracilis muscle as the donor muscle. Patientswith traumatic muscle loss and those who received FFMTusing muscles other than the gracilis as the donor musclewere excluded. At the beginning of the study period, weused traditional end-to-end vascular anastomosis whenperforming FFMT, whereas in the later part of the studyperiod, we used the flow-through technique; thus, patientswere divided into a traditional anastomosis group and aflow-through anastomosis group.Data regarding patient age and gender, the etiology of the

injury, the harvest time, the total operation time, vascularcompromise and other postoperative complications, anddonor-site complications were collected from the medicalrecords. The data were specifically collected from themedical records by a physician who was not involved inthe study and who was not aware of which type ofanastomosis was performed. As is routine in our department,two groups of surgeons began preparation of the donor siteand recipient site simultaneously. The operation time wasdefined as the time from incision to wound closure, and the

harvest time was defined as the time from incision tocomplete isolation of the muscle. The study was approved bythe Institutional Review Board of the hospital, and allpatients provided written informed consent for the surgicalprocedure performed and for their images and data to beused for research purposes.

Surgical techniqueAll of the FFMTs were performed by a single senior

professor and his team, including a resident and an attendingphysician. Each patient was placed in the supine positionwith hip joint flexion, abduction, and external rotation andknee joint flexion. In this position, the adductor longus couldbe palpated in the medial thigh. Additionally, a line wasdrawn along the prominence from the pubic tubercle to themedial knee to indicate the anterior boarder of the gracilismuscle, and the skin flap overlying the gracilis muscle wasmarked (Figure 1).An incision was made along the anterior boarder of the

skin flap, and dissection between the adductor longus andthe gracilis was performed, preserving the fascia surroundingthe gracilis. The dominant pedicle and motor nerve wereidentified beneath the adductor longus (Figure 2A); however,the neurovascular pedicle was not dissected at this time.Next, the incision was extended to the insertion of the gracilis,and the posterior border of the skin flap was incised. Thegracilis was then isolated from adductor longus anterolat-erally and from the adductor magus posterolaterally.With the adductor longus retracted anteriorly, the vascular

pedicle of the gracilis was exposed and dissected, and thebranches to the adductor longus, brevis, and magnus wereligated. The nerve branches of the muscle were also identified(Figure 2B). The adductor longus was also retracted poster-omedially so that the artery pedicle could be traced to itsorigin at the profunda femoris. A 2 cm to 3 cm segment ofthe profunda femoris was then isolated and cut (Figure 2C).A T-shaped artery pedicle comprising the profunda femorisand the nutrient artery of the gracilis was harvested, and thevenae comitantes of the nutrient artery were also harvestedfrom their origin. The profunda femoris arterial segment hadan obviously larger caliber (Figure 2D). After dividing themotor nerve, the gracilis was harvested.Incisions in the upper limb were performed based on the

aim of reconstruction. The recipient artery, which was usuallythe brachial artery, axillary artery, or radial artery, was dividedand interposed with the T-shaped pedicle, with both endsanastomosed (Figure 3). The venae comitantes of the graciliswere also anastomosed with the matched recipient veins in anend-to-end fashion. If there were 2 venae comitantes, theywere anastomosed with the superficial and deep venoussystems, respectively (Figure 3C). When only a single venacomitans was present, only one anastomosis was performed.In the end-to-end anastomosis group, a single arterial end-to-end anastomosis was performed. The spinal accessory nervewas the first choice for nerve innervation in both groups. Forpatients whose spinal accessory nerves were unavailable,intercostal nerves, the medial brachial cutaneous nerve, and abundle of ulnar nerves reinnervated by CC7 were utilized.

Postoperative care and follow-upPostoperatively, the patients were administered antibiotics

to prevent infection and were also given anticoagulationand anticonvulsant treatments. Plaster splints were applied for

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6 weeks after surgery, and passive functional exercises werebegun immediately after surgery. The rehabilitation protocolcomprised acupuncture, moxibustion, and electrical stimulation.

Donor-site discomfort and dysfunction were evaluated 1year after the FFMT. Subjective donor-site discomfort wasspecifically evaluated using a questionnaire adapted from

Figure 1 - Design of the gracilis musculocutaneous flap

Figure 2 - Intraoperative images - A) Exposure of the dominant vascular pedicle. To avoid injury during the operation, the pedicle wasnot dissected at first. B) The neurovascular pedicle of the gracilis. Note that the sensory nerve branch ($) must be resected to ensureenough motor nerve fiber regeneration (a, b). C) Exposure of the profunda femoris. A segment of the profunda femoris was prepared.It is unnecessary to perform a long dissection. D) The T-shaped arterial pedicle of the gracilis musculocutaneous flap (flap placed withthe skin paddle downward).

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that of Carr et al. (31). Briefly, donor-site symptoms wereclassified into 11 grades, with 0 defined as no discomfort; 1 to9, as increasing degrees of discomfort; and 10, as unbearablediscomfort.

Statistical analysisContinuous variables are presented as the mean±stan-

dard deviation (SD) and were compared using independent-sample t-tests. Categorical variables are expressed as anumber and percentage and were compared using Fisher’sexact test. A two-tailed po0.05 was considered statisticallysignificant. All analyses were performed using SPSS Version20 (SPSS Statistics V20, IBM Corporation, Somers, NewYork).

’ RESULTS

A total of 71 patients treated with FFMT for traumatic BPIdue to a motorcycle accident, a machine injury, or a crushinjury were included in the analysis. A total of 46 patientsreceived flow-through anastomosis, and 25 patients receivedconventional end-to-end anastomosis; these 2 groups werecomparable with respect to age and sex (both p40.05;Table 1). The purpose of reconstruction for the patients in theflow-through group was to restore elbow flexion and fingerextension (n=33), to restore elbow flexion and finger flexion(n=12), or to restore elbow extension (n=1). In the end-to-endgroup, the goal of reconstruction was to restore elbow flexionand finger extension (n=21) or to restore elbow flexion (n=4).In the flow-through group, the recipient vessels included

32 brachial arteries, five axillary arteries, and nine radialarteries. In the end-to-end group, the recipient vesselsincluded four circumflex humeral arteries, six brachial arterybranches, six axillary artery branches, three deep brachialartery branches, and two thoracoacromial arteries.As shown in Table 1, the harvest time (p=0.167), total

operation time (p=0.721), and donor-site score (p=0.288) weresimilar between the two groups. The diameter of the arterialanastomosis in the flow-through group was significantlylarger than that in the end-to-end group (3.87±0.42 mm vs.2.06±0.44 mm, respectively, po0.001), and there weresignificantly fewer cases of vascular compromise in theflow-through group (2 [4.35%] vs. 6 [24%], respectively,p=0.019). Moreover, in the flow-through group, there weretwo cases of reversible venous spasms, and the flaps weresalvaged after re-exploration. In contrast, in the end-to-endgroup, there were six cases of vascular compromise,including one case of venous spasm, four of venousthrombosis, and one of arterial thrombosis; four flaps weresalvaged, whereas two failed.In addition, one patient in the flow-through group and

two in the end-to-end group developed donor-site hemato-mas that resolved with local care. However, no adductionmuscle strength loss was noted at 1 year after surgery ineither group, as determined by a manual muscle strengthtest. The donor-site scores were similar between the twogroups (Table 1), and no scores of 3 or more were noted ineither group. Discomfort included itching, numbness, andhyperesthesia around the scar. However, all discomfort waseasily bearable and did not affect the activities of daily life.

Figure 3 - Flow-through anastomosis of the T-shaped pedicle - A) The diameter of the profunda femoris segment is obviously largerthan that of the nutrient artery of the gracilis. B) The brachial artery was resected, and the diameters of the segment profunda femorisand brachial artery were well matched. C) Interposed anastomosis to bridge the brachial artery. Two veins were anastomosed in directend-to-end fashion.

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’ DISCUSSION

We have performed FFMT for brachial plexus avulsionsince 2005, and end-to-end vascular anastomosis was initiallyused. As the technique evolved, we adopted flow-throughvascular anastomosis for the procedure. In the present study,we compared the results of gracilis FFMT for traumatic BPIusing flow-through or end-to-end anastomosis. The resultsshowed that the diameter of the arterial anastomosis in theflow-through group was significantly larger than that in theend-to-end group. Additionally, there were significantly fewercases of vascular compromise and less flap failure in the flow-through anastomosis group, whereas the operation time anddonor-site morbidity were similar between the groups.The treatment of BPI is challenging, especially if there is

complete avulsion of the brachial plexus. Limited donornerve for nerve transfer and long distances to the targetmuscle are the main obstacles. For certain patients, FFMT isthe only choice for the restoration of limb function. In thesecases, FFMT failure would be catastrophic. The reportedsuccess rate of free tissue transplantation ranges from 91% to99%, with the majority of failures being due to technicalerrors with vessel anastomosis, such as a mismatch of vessels(16). Generally, small vessels are more vulnerable tothrombosis than large-diameter vessels are, and a T-shapedvascular pedicle can afford a larger vessel for anastomosis.Although primarily used in trauma with main artery injury,

application of the flow-through technique has been extendedto various situations without vascular injury. For example,Haffey et al. (25) reported a flow-through anterolateral thighfree flap that could capture vascular perforators from separatesources, regardless of the vascular branching pattern ofthe pedicle. Moreover, Kawamura et al. (27) reported thefeasibility of harvesting a flow-through flap from the scapularregion, and Koshima et al. (28) reported a flow-through freeanterolateral or anteromedial thigh flap with a wide but shortvascular pedicle to avoid problems associated with variationand to shorten the operation time, with good results. Thus, theversatility of the technique has been clearly shown, layingthe foundation for its application in FFMT.The gracilis muscle has a type II blood supply system (32).

The dominant pedicle enters the muscle 6 to 10 cm inferior tothe pubic tubercle, with one or two minor pedicles enteringthe muscle approximately 20 cm inferior to the pubis. Themuscle can survive by relying only on the dominant vascularpedicle, and the caliber of the dominant pedicle is 1 to 2.5 mm(17). The dominant artery generally originates from theprofunda femoris (33), although certain authors havereported that it originates from the medial circumflex femoralartery (34). Regardless of the origin, both the profunda

femoris and the medial circumflex femoral artery can beeasily identified and well matched with a recipient artery.

The most important advantage of a T-shaped vascularpedicle is the large caliber, and there are unique advantagesto using this technique in FFMT. Miyamoto et al. (35)revealed that a flow-through flap allowed greater blood flowinto the flap through the anastomotic site than end-to-endand end-to-side anastomoses did. For FFMT, increased bloodflow is particularly advantageous for intramuscular bloodcirculation during the early stage after surgery. Extradissection to identify an anonymous recipient artery isunnecessary because of matching with the brachial, axillary,or radial artery. This approach successfully avoids problemsdue to previous vascular injury and also allows for variationof the insertion point of the transplanted muscle, withoutrestriction due to inconvenient recipient vessel positioning.Additionally, the vascular system of the recipient site can bekept intact, which reduces the risk of additional ischemia ifrecipient vessels must be sacrificed.

There are, however, several unresolved issues with theapplication of a T-shaped venous pedicle. Ichinose et al. (36)noted that dual venous anastomosis of separate venoussystems is conducive to reducing the risk of flap failure andaffords protection against venous catastrophe through a self-compensating mechanism. There are one or two venaecomitantes in the gracilis vascular pedicle, and with flow-through venous anastomosis, drainage occurs through asingle venous system. Therefore, when there are two venaecomitantes, it is recommended that they be respectivelyanastomosed with branches from the deep and superficialvenous systems. Conversely, if only one vena comitans ispresent, flow-through venous anastomosis is recommendeddue to the advantages of the large caliber.

In the present study, less vascular compromise wasobserved with flow-through anastomosis; the increasedinflow not only supplies oxygen-rich blood but alsoimproves the outflow of venous blood (35). This is likelythe reason that significantly less vascular crisis occurred inthe flow-through group, even though only the arteryunderwent flow-through anastomosis. Due to abundantcommunication in the femoral arterial system in the thigh,ischemia of the lower limb after sacrifice of the profundafemoris is rare. In our study, no ischemic necrosis orcontracture of the muscles at the donor site occurred, andno obvious decrease in muscle strength was observed.

There are no specific contraindications for the use of flow-through anastomosis with a T-shaped arterial pedicle inFFMT. However, caution must be exercised in patients with ahistory of vascular injury to the femoral artery. Computedtomography angiography (CTA) may be useful for assessing

Table 1 - Patient characteristics and outcomes

Flow-through group (n=46) End-to-end anastomosis group (n=25) p-value

Age (y) 27.85±8.80 26.44±7.94 0.508Gender, male 44 (95.6) 24 (96) 0.718Harvest time (min) 98.67±9.16 101.52±6.02 0.167Total operation time (min) 400.11±73.14 408.20±98.74 0.721Diameter of arterial anastomosis* (mm) 3.87±0.42 2.06±0.44 o0.001Vascular compromise 2 (4.35) 6 (24) 0.019Donor-site score 1.09±0.69 0.88±0.67 0.228

* The diameter was measured once the vessels were mobilized during surgery.The data are expressed as a number (percentage) or the mean±standard deviation.

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blood flow in the femoral artery system preoperatively. Inaddition, the method is not recommended if the ipsilateralgracilis is chosen as the donor muscle; due to the relativelocations of the donor and recipient vessels, folding andtwisting of the vascular pedicle is likely to occur.The primary limitations of this report are the relatively small

number of patients and the retrospective nature of the study.Traumatic muscle loss is also an important indication for FFMT;however, we limited this study to patients without muscle lossto reduce the influence of this variable on the results. Lastly, wedid not include data on the long-term outcomes of the surgerybecause the purpose of this report was to examine the effect ofthe flow-through technique on flap survival.The use of flow-through anastomosis in gracilis FFMT for

BPI can decrease the complexity of anastomosis and reducethe risk of flap loss. The technique allows for a recipientartery to be chosen such that muscle placement can beperformed in accordance with the aims of reconstruction andalso allows for preservation of the original vessels in therecipient limb. Thus, we believe that flow-through anasto-mosis should be considered when performing FFMT.

’ ACKNOWLEDGMENTS

This study was supported by the NHFPC Special Fund for Health ScientificResearch in the Public Welfare (Number 201402016).


We declare that all of the listed authors have participated actively in thestudy and meet the requirements for authorship. Hou Y and Gu L designedthe study and wrote the protocol. Hou Y and Yang J performed theresearch/study. Liu X and Zhu Q contributed constructive suggestionsabout writing the article. Yang Y and Qin B managed the literaturesearches and analyses. Fu G and Li X performed the statistical analysis.Hou Y wrote the first draft of the manuscript.

’ REFERENCES

1. Midha R. Epidemiology of brachial plexus injuries in a multitraumapopulation. Neurosurgery. 1997;40(6):1182-9, http://dx.doi.org/10.1097/00006123-199706000-00014

2. Gu YD, WU MM, Zhen YL, Zhao JA, Zhang GM, Chen DS, et al. Phrenicnerve transferfor treatment of root avulsion of the brachial plexus. ChinMed J (Engl). 1990;103(4):267-70.

3. Mcguiness CN, Kay SP. The prespinal route in contralateral C7 nerve roottransfer for brachial plexus avulsion injuries. J Hand Surg Br. 2002;27(2):159-60, http://dx.doi.org/10.1054/jhsb.2001.0665

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Treatment with dasatinib or nilotinib in chronic myeloidleukemia patients who failed to respond to twopreviously administered tyrosine kinase inhibitors –a single center experienceBeatriz Felicio Ribeiro, Eliana C.M. Miranda, Dulcineia Martins de Albuquerque, Marcia T. Delamain,

Gislaine Oliveira-Duarte, Maria Helena Almeida, Bruna Vergılio, Rosana Antunes da Silveira,

Vagner Oliveira-Duarte, Irene Lorand-Metze, Carmino A. De Souza, Katia B.B. Pagnano*

Universidade de Campinas (Unicamp), Centro de Hematologia e Hemoterapia Campinas/SP, Brazil.

OBJECTIVE: To evaluate hematological, cytogenetic and molecular responses as well as the overall, progression-free and event-free survivals of chronic myeloid leukemia patients treated with a third tyrosine kinase inhibitorafter failing to respond to imatinib and nilotinib/dasatinib.

METHODS: Bone marrow karyotyping and real-time quantitative polymerase chain reaction were performed atbaseline and at 3, 6, 12 and 18 months after the initiation of treatment with a third tyrosine kinase inhibitor.Hematologic, cytogenetic and molecular responses were defined according to the European LeukemiaNetrecommendations. BCR-ABL1 mutations were analyzed by Sanger sequencing.

RESULTS: We evaluated 25 chronic myeloid leukemia patients who had been previously treated with imatinib and asecond tyrosine kinase inhibitor. Nine patients were switched to dasatinib, and 16 patients were switched to nilotinibas a third-line therapy. Of the chronic phase patients (n=18), 89% achieved a complete hematologic response, 13%achieved a complete cytogenetic response and 24% achieved a major molecular response. The following BCR-ABL1mutations were detected in 6/14 (43%) chronic phase patients: E255V, Y253H, M244V, F317L (2) and F359V. M351Tmutation was found in one patient in the accelerated phase of the disease. The five-year overall, progression-free andevent-free survivals were 86, 54 and 22% (po0.0001), respectively, for chronic phase patients and 66%, 66% and 0%(po0.0001), respectively, for accelerated phase patients. All blast crisis patients died within 6 months of treatment.Fifty-six percent of the chronic phase patients lost their hematologic response within a median of 23 months.

CONCLUSIONS: Although the responses achieved by the third tyrosine kinase inhibitor were not sustainable, athird tyrosine kinase inhibitor may be an option for improving patient status until a donor becomes availablefor transplant. Because the long-term outcome for these patients is poor, the development of new therapies forresistant chronic myeloid leukemia patients is necessary.

KEYWORDS: CML; Dasatinib; Nilotinib; Third-line TKI treatment.

Ribeiro BF, Miranda EC, Martins de Albuquerque D, Delamain MT, Oliveira-Duarte G, Almeida MH, et al. Treatment with dasatinib or nilotinib inchronic myeloid leukemia patients who failed to respond to two previously administered tyrosine kinase inhibitors – a singlecenter experience. Clinics. 2015;70(8):550-555

Received for publication on February 25, 2015; First review completed on March 30, 2015; Accepted for publication on May 21, 2015



’ INTRODUCTION

Second-generation tyrosine kinase inhibitors (TKIs),such as nilotinib and dasatinib, are effective therapeuticoptions for chronic myeloid leukemia (CML) patients who

have failed to respond to imatinib as a first-line therapy.Indeed, approximately 50% of chronic phase (CP) patientsachieve a complete cytogenetic response (CCyR) whentreated with second-generation TKIs (1,2). Nevertheless,approximately 52% of patients must discontinue second-line TKI therapy, most often due to resistance or intoler-ance (3).

Allogeneic transplantation is the treatment of choice forpatients who fail to respond to at least one second-generationTKI (2nd TKI). However, transplantation is not feasible formany older patients, for patients with poor performancestatus and for patients who do not have an available donor.DOI: 10.6061/clinics/2015(08)04

Copyright & 2015 CLINICS – This is an Open Access article distributed under theterms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0/)which permits unrestricted use, distribution, and reproduction in any medium orformat, provided the original work is properly cited.


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CLINICAL SCIENCE

These patients may be switched to a different TKI that wasnot used previously or switched to other drugs, such asinterferon (INF) or hydroxyurea (HU) (4). Patients treatedwith a 3rd TKI should be closely monitored because novelmutations can occur with sequential TKI therapy, increasingthe risk of resistance (5,6).In Brazil, fewer treatment options are available for

resistant cases as other TKIs, such as bosutinib andponatinib, are not available. In this study, we present ourexperience with CML patients treated with dasatinib ornilotinib as a third-line (3rd TKI) therapy and emphasize theimportance of developing other therapeutic options for thesepatients.

’ PATIENTS AND METHODS

Between July 2008 and December 2014, 213 CML patientswere treated at the Hematology and Hemotherapy Center atthe University of Campinas according to the 2006 and 2009European LeukemiaNet recommendations (7,8). The first-line treatment for CML in Brazil is imatinib. The second-lineTKI is chosen based on clinical factors, BCR-ABL1 mutationstatus and drug availability. Dasatinib was approved in 2008and nilotinib was approved in 2009 in Brazil; before 2008,these drugs were available only through clinical trials. Atotal of 25 consecutive adult CML patients, 18 (72%) ofwhom were in the CP stage, 3 (12%) of whom were in the APstage and 4 (16%) of whom were in the BC stage, who wereresistant (n=23) or intolerant (n=2) to two prior TKIs andwere switched to a 3rd TKI, were included in our analysis.Most of the patients were treated at our center since theirinitial diagnosis; however, three patients were referred fromother treatment centers at the time of initiation of the 2nd TKItreatment and were followed at our center after discontinua-tion of the 2nd TKI. Patients were treated with 100-140 mgdasatinib daily (n=9) (after failure with imatinib andnilotinib) or 400-800 mg nilotinib daily (n=16) (after failurewith imatinib and dasatinib). Doses were adjusted accordingto tolerance. Hematologic, cytogenetic and molecularresponses as well as the CML phases were defined accordingto the European LeukemiaNet recommendations (8,9). Bonemarrow karyotyping was performed using the Giemsa-Trypsin-Wright stain banding technique at baseline and at3, 6, 12 and 18 months after the initiation of therapy with the3rd TKI. Twenty metaphase cells were analyzed for eachsample (10).

Detection of BCR-ABL1 transcriptsBCR-ABL1 transcripts were measured in the peripheral

blood by real-time quantitative polymerase chain reaction(RQ-PCR) at baseline and then every 3 months usingprocedures described elsewhere with some modifications(11). First, cDNAwas amplified using the ABI 7300 sequencedetection system (Applied Biosystems) and TAQMANUniversal Master Mix in a final reaction volume of 25 mLaccording to the instructions recommended by the manu-facturer. ABL1 was used for normalization. BCR-ABL1transcripts were measured in duplicate. The copy numberswere calculated by comparison with a standard curvegenerated from serial dilutions (4-6 dilutions) of a linearizedplasmid containing a BCR-ABL1 insert, which has beendescribed previously (12). The results were reported as BCR-ABL1/ABL1 ratio (%) after conversion to the international

scale (IS). Major molecular response (MMR) was defined as atranscript level p0.1% (IS).

Detection of BCR-ABL1 kinase domain mutationsMutations were detected by direct sequencing of DNA

from peripheral blood samples collected from TKI-resistantCML patients who failed or displayed a sub-optimalresponse to IM or a 2nd TKI, according to methods thatwere described previously (13,14). Briefly, total RNA wastranscribed to cDNA and then was amplified using Taqplatinum high fidelity and primers; the forward primerannealed to BCR exon 2, and the reverse primer annealed toABL exon 10. The PCR product was amplified in a semi-nested reaction, resulting in a 863-base pair fragment thatwas sequenced in both directions. The sample nucleotidesequences were compared to the GenBank accessionno. X16416.

Statistical methodsProbabilities of overall survival (OS), progression-free

survival (PFS) and event-free survival (EFS) were calculatedusing the Kaplan-Meier method. OS was calculated at theinitiation of therapy with the 3rd TKI until the final follow-upor death for any reason. PFS was defined as survival withouttransformation to the accelerated or blastic phase afterstarting the 3rd TKI and was judged based on an event ofprogression or death. EFS was defined as loss of completehematological response (CHR), CCyR, MMR, progression toadvanced phases, death or 3rd TKI discontinuation for anyreason (toxicity, resistance, transplant or patient lost tofollow-up). Po0.05 was considered statistically significant.The cut-off for the data analysis was March 2015.

EthicsThe study protocol was approved and was conducted in

accordance with the ethical standards of the local ResearchEthics Committee on human experimentation and theHelsinki Declaration of 1975, which was revised in 1983.Patients provided written informed consent for theirparticipation.

’ RESULTS

Clinical and laboratory characteristics of the 25 CMLpatients at the time of diagnosis and before the initiation ofthe 3rd TKI are presented in Tables 1 and 2, respectively.Chronic-phase CML patients (CP-CML) (n=18) were

analyzed separately. Thirteen CP-CML patients were resis-tant to imatinib (72%), and 5 were intolerant to imatinib(28%). Five patients were treated with dasatinib (28%), and13 patients were treated with nilotinib (72%). Sixteen patients(89%) were resistant to the 2nd TKI, and 2 patients (11%) wereintolerant to the 2nd TKI. The resistant patients neverachieved a previous CCyR with imatinib or with the 2nd

TKI. The median follow-up duration was 52 (7-75) months,and 16/18 patients (89%) achieved or maintained a completehematologic response during this period. Of 15 patients whowere subjected to cytogenetic analysis, 2 (13%) achievedCCyR. Of 17 CP-CML patients with available molecularanalysis data, 4 (24%) achieved a major molecular response(MMR), and 2 achieved a complete molecular response(CMR). For CP-CML patients, the frequencies of the transcriptlevels at baseline and at 3 and 6 months after the initiation ofthe 3rd TKI are shown in Table 3.

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CLINICS 2015;70(8):550-555 CML patients treated with a third TKIRibeiro BF et al.

Mutation analysisBCR-ABL1 mutations were evaluated in 14 of 18 CP-CML

patients, and mutations were detected in 6/14 patients(43%). One patient in the AP stage presented with themutation M351T. The mutation F317L was found in 3patients before the initiation of the 3rd TKI (during second-line dasatinib therapy), and the mutation F359V was foundin one patient, who displayed imatinib resistance, before theinitiation of dasatinib as a 2nd-line therapy. Five mutationswere found during 3rd-line TKI therapy: E255V (dasatinib),Y253H (dasatinib), M244V (dasatinib), and F317L (nilotinib).The patient with the F359V mutation presented with a longhistory of disease and had been treated previously for12 years at another center with busulfan and hydrea beforeimatinib treatment. The F359V mutation was detected for thefirst time when a patient developed imatinib resistance, butat the time there were no 2nd-line inhibitors available inBrazil. The patient underwent hematopoietic stem celltransplantation (HSCT) and relapsed one and a half yearslater with persistence of the F359V mutation. The patient wastreated with dasatinib and achieved CHR but never achieveda major cytogenetic response. After 4 years of dasatinibtreatment, the patient progressed to the AP stage. At thistime, a new mutation analysis was performed, whichrevealed no evidence of the F359V mutation, but a newmutation, F317L, was identified. The patient was treatedwith nilotinib and achieved CHR but relapsed after5 months; at the time of relapse, the patient maintained the

F317L mutation. A second HSCT was performed, whichresulted in the achievement of a complete molecular response,but the patient died due to graft-versus-host disease.

Survival analysisOne patient in the CP stage died during 3rd TKI therapy.

CP-CML patients had 5-year OS, PFS and EFS values of 86,54 and 22% (po0.0001), respectively, whereas AP-CMLpatients had 5-year OS, PFS and EFS values of 66, 66 and0%, respectively (po0.0001). BC-CML patients showed noresponse in the first year after treatment (Figures 1, 2 and 3).

Long-term outcomeDuring treatment, 9/16 (56%) CP-CML patients lost CHR

within a median of 23 (3-37) months. Two patients lost CCyRafter 12 and 13 months. One patient lost MMR after7 months. Six (34%) patients are currently taking their 3rd

TKI, although 3 of these patients lost their response (1 MMR,1 CCyR and 1 CHR). Three CP-CML patients (17%)progressed to the BC (blast crisis) stage, and 2 CP-CMLpatients subsequently died. Discontinuation of the 3rd TKIoccurred in 16 (89%) cases due to resistance (8); intolerance(3); loss to follow-up (3); and death (2) during the treatment.

Three AP-CML patients reached CHR, but one of thesepatients lost their response. Only one patient achieved CCyRand MMR, but those responses were lost. One patientdiscontinued treatment due to intolerance in the 4th month.

Table 1 - Characteristics of chronic myeloid leukemia patients at diagnosis (n=25).

Variables n. %

Median age (range) years 45 (14-72)Gender: male 13 52Sokal risk groupLow 5 20Intermediate 1 4High 9 36Missing 10 40

Additional chromosomal abnormalities* 01/09 11.1Splenomegaly 11/16 68.7Spleen size 410 cm below the costal margin 06/11 54.4White cell count x 109/L (median, range) 137.10 (17.1 – 494.4)Platelet count x 109/L (median, range) 352.0 (141.0 - 2,901.0)Hemoglobin, g/L (median, range) 10.2 (5.1 – 13.7)Blasts PB, % (median, range) 3.5 (0 - 17)Basophils PB, % (median, range) 4 (0 - 34)

* 47, XX, t (9;22) (q34;q11), +der(22)

Table 2 - Clinical and laboratory characteristics of chronicmyeloid leukemia patients at the initiation of the 3rd tyrosinekinase inhibitor (n=25).

Variables n= 25

Median age (range) years 56 (22-75)Median time of imatinib therapy (range) months 30 (1-66)Achievement of CCyR with imatinib treatment n (%) 3 (12%)Interval diagnosis – 3rd TKI (range) months 98 (12-404)Treated with dasatinib 100-140 mg once daily n (%) 16 (64%)Treated with nilotinib 400 mg BID n (%) 09 (36%)Disease status before 3rd TKI n (%)

CP 18 (72%)AP 03 (12%)BC 04 (16%)

Table 3 - Molecular responses of chronic phase-chronic myeloidleukemia patients treated with a 3rd tyrosine kinase inhibitor.

Time RQ-PCR (IS)% N %

Baseline 410 11/18 611 – 10 04/18 22

0.1 – 1o 03/18 17p0.1 0 0

3 months 410 09/12 751 – 10 02/12 17

0.1 – 1o 0 0p0.1 1/12 08

6 months 410 04/08 501 – 10 01/08 12.5

0.1 – 1o 01/08 12.5p0.1 02/08 25

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CML patients treated with a third TKIRibeiro BF et al.

CLINICS 2015;70(8):550-555

Figure 1 - Kaplan-Meier survival analysis. Five-year OS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitoraccording to disease phase.

Figure 2 - Kaplan-Meier survival analysis. Five-year PFS of chronic myeloid leukemia patients treated with a 3rd tyrosine kinase inhibitoraccording to disease phase.

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Four BC-CML patients did not reach hematological orcytogenetic responses and died within four months of theinitiation of the 3rd TKI.Regarding other treatments after discontinuation of the 3rd

TKI, 14 patients were treated with the following drugs:hydrea (8), hydrea followed by HSCT (2), hydrea followedby low dose ARA-C and imatinib (1), interferon followed byhydrea (1), imatinib (1), and conventional chemotherapyfollowed by hydrea (1).

’ DISCUSSION

Our data show that only 22% of patients in the CP stageshowed long-term benefits from the administration of a 3rd

TKI after imatinib and a 2nd TKI failure. We found that 89%of our patients in the CP stage achieved CHR, 13% achievedCCyR, and 24% achieved MMR; however, 50% of thosepatients lost CHR within a median of 23 months. All patientswith CCyR lost their response after 12 months, and 25% ofpatients lost MMR after 7 months.Our results are in agreement with prior reports. Quintas-

Cardama et al. (15) performed a study on 23 CML patientstreated with dasatinib after imatinib and nilotinib failure andfound that 43% of these patients achieved CHR and 30%achieved a cytogenetic response. Giles et al. (16), performed astudy analyzing 60 patients treated with nilotinib afterimatinib and dasatinib failure and found that 70% of CP-CML patients achieved CHR and 43% of CP-CML patientsachieved a major cytogenetic response (MCyR). The authorsalso found that after 18 months, 59% of CP-CML patientswere progression-free, and their estimated survival was 86%.

Regarding molecular responses, most of our patients hadBCR-ABL1 transcript levels 410% at 3 months (75%) and41% at 6 months (62.5%). The achievement of earlyresponses to first- and second-line therapies, such as BCR-ABL1 transcript levels o10% at 3 months and o1% at6 months, has been associated with long-term cytogeneticand molecular responses and better clinical outcomes(3,17–22). Only one patient in the CP stage achieved theoptimal response criteria within 3 months and 6 months,respectively.

In our study, the EFS was 44% at 27 months for CP-CMLpatients, which is similar to the findings reported by Ibrahimet al. (23), where 26 CP-CML patients who failed to respondto two prior TKIs had 45.7% EFS at 30 months after theinitiation of a 3rd TKI. These results show that althoughpatients can achieve hematological and cytogeneticresponses with a 3rd TKI, those responses are not sustainable.Similar observations were made by Garg et al.(24). Theauthors evaluated 48 CML patients, 25 of whom were inthe CP stage, treated sequentially with three TKIs. Threepatients in the CP stage and one in the AP stage achievedCCyR; the median duration of the response was 16.3 months.The median failure-free survival was 20 months for patientsin the CP stage, 5 months for patients in the AP stage, and3 months for patients in the BP stage.

Patients treated with sequential TKIs also have a higherrisk of developing resistance and novel mutations (5,6). Infact, 76% of our patients discontinued the 3rd TKI, and 42%of those discontinuations were due to resistance. Wefound 5 BCR-ABL1 mutations in 14 CP patients during the3rd TKI therapy. One patient harbored a F359V mutationand responded to dasatinib, however, another mutation

Figure 3 - Kaplan-Meier survival analysis. Five-year event-free survival of chronic myeloid leukemia patients treated with a 3rd tyrosinekinase inhibitor according to disease phase.

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CML patients treated with a third TKIRibeiro BF et al.

CLINICS 2015;70(8):550-555

was selected in this patient when the disease progressed(F317L).Although the responses to 3rd-line TKI therapy are not

sustainable, 3rd-line TKIs may be an alternative for patientswith CML who failed to respond to imatinib and a secondgeneration TKI and are not eligible for HSCT (4). A 3rd-lineTKI can improve the patient’s condition until an alternativetransplant donor is available. Nevertheless, because the long-term outcome of these patients is poor, it is important toemphasize the importance of developing new therapies forCML resistant patients.

’ ACKNOWLEDGEMENTS

Beatriz Felicio Ribeiro received a scholarship from FAPESP and KatiaPagnano received financial support from FAPESP. The authors thank theUniversidade de Campinas (UNICAMP), Centro de Hematologia eHemotherapia, Campinas/SP, Brazil for supporting this study.


Ribeiro BF and Pagnano KB conceived and designed the study. RibeiroBF, Duarte VO, Miranda EC, Almeida MH, and Pagnano KB performedthe data collection. Delamain MT, Oliveira-Duarte G, and Pagnano KBtreated the patients. Lorand-Metze I, Souza CA, Pagnano KB, Ribeiro BF,Vergílio B, Silveira RA, and Albuquerque DM performed the BCR-ABL1mutation analysis and quantitative PCR experiments. Miranda ECMmanaged, analyzed, and interpreted the data. Ribeiro BF, Miranda ECM,Albuquerque DM, Delamain MT, Oliveira- Duarte G, Almeida MH,Vergílio B, Silveira RA, Oliveira-Duarte V, Lorand-Metze I, Souza CA,and Pagnano KB approved the final manuscript. All authors contributed tothe collection, analysis and interpretation of the data and contributed to thecritical revision of the article for intellectual content.

’ REFERENCES

1. Giles FJ, Le Coutre PD, Pinilla-Ibarz J, Larson RA, Gattermann N,Ottmann OG, et al. Nilotinib in imatinib-resistant or imatinib-intolerantpatients with chronic myeloid leukemia in chronic phase: 48-monthfollow-up results of a phase II study. Leukemia. 2013;27(1):107–12,http://dx.doi.org/10.1038/leu.2012.181.

2. Hochhaus A, Baccarani M, Deininger M, Apperley JF, Lipton JH,Goldberg SL, et al. Dasatinib induces durable cytogenetic responses inpatients with chronic myelogenous leukemia in chronic phase withresistance or intolerance to imatinib. Leukemia. 2008;22(6):1200–6, http://dx.doi.org/10.1038/leu.2008.84.

3. Milojkovic D, Apperley JF, Gerrard G, Ibrahim AR, Szydlo R, Bua M,et al. Responses to second-line tyrosine kinase inhibitors are durable: anintention-to-treat analysis in chronic myeloid leukemia patients. Blood.2012;119(8):1838–43, http://dx.doi.org/10.1182/blood-2011-10-383000.

4. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, ApperleyJF, et al. European LeukemiaNet recommendations for the management ofchronic myeloid leukemia: 2013. Blood. 2013;122(6):872–84, http://dx.doi.org/10.1182/blood-2013-05-501569.

5. Hughes T, Saglio G, Branford S, Soverini S, Kim D-W, Müller MC, et al.Impact of baseline BCR-ABL mutations on response to nilotinib inpatients with chronic myeloid leukemia in chronic phase. J Clin Oncol.2009;27(25):4204–10, http://dx.doi.org/10.1200/JCO.2009.21.8230.

6. Cortes J, Jabbour E, Kantarjian H, Yin CC, Shan J, Brien SO, et al. Dynamicsof BCR-ABL kinase domain mutations in chronic myeloid leukemia aftersequential treatment with multiple tyrosine kinase inhibitors. Blood.2007;110(12):4005–11, http://dx.doi.org/10.1182/blood-2007-03-080838.

7. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B,Appelbaum F, et al. Evolving concepts in the management of chronicmyeloid leukemia: recommendations from an expert panel on behalf ofthe European LeukemiaNet. Blood. 2006;108(6):1809–20, http://dx.doi.org/10.1182/blood-2006-02-005686.

8. Baccarani M, Cortes J, Pane F, Niederwieser D, Saglio G, Apperley J, et al.Chronic myeloid leukemia: an update of concepts and managementrecommendations of European LeukemiaNet. J Clin Oncol. 2009;27(35):6041–51, http://dx.doi.org/10.1200/JCO.2009.25.0779.

9. Cortes JE, Talpaz M, O’Brien S, Faderl S, Garcia-Manero G, Ferrajoli A,et al. Staging of chronic myeloid leukemia in the imatinib era: anevaluation of the World Health Organization proposal. Cancer. 2006: 106(6):1306-15, http://dx.doi.org/10.1002/(ISSN)1097-0142.

10. Testoni N, Marzocchi G, Luatti S, Amabile M, Baldazzi C, Stacchini M,et al. Chronic myeloid leukemia: a prospective comparison of interphasefluorescence in situ hybridization and chromosome banding analysis forthe definition of complete cytogenetic response: a study of the GIMEMACMLWP. Blood. 2009;114(24):4939–43, http://dx.doi.org/10.1182/blood-2009-07-229864.

11. Machado MP, Tomaz JP, Lorand-Metze I, Souza CA De, Vigorito AC,Delamain MT, et al. Monitoring of BCR-ABL levels in chronic myeloidleukemia patients treated with imatinib in the chronic phase – theimportance of a major molecular response. Rev Bras Hematol Hemoter.2011;33(3):211–5, http://dx.doi.org/10.5581/1516-8484.20110056.

12. Cross NC, Feng L, Chase A, Bungey J, Hughes TP, Goldman JM. Com-petitive polymerase chain reaction to estimate the number of BCR-ABLtranscripts in chronic myeloid leukemia patients after bone marrowtransplantation. Blood. 1993;82(6):1929–36.

13. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, et al.Monitoring CML patients responding to treatment with tyrosine kinaseinhibitors: review and recommendations for harmonizing current method-ology for detecting BCR-ABL transcripts and kinase domain mutationsand for expressing results. Blood. 2006;108(1):28–37, http://dx.doi.org/10.1182/blood-2006-01-0092.

14. Branford S, Rudzki Z, Walsh S, Parkinson I, Grigg A, Szer J, et al.Detection of BCR-ABL mutations in patients with CML treated withimatinib is virtually always accompanied by clinical resistance, andmutations in the ATP phosphate-binding loop (P-loop) are associatedwith a poor prognosis. Blood. 2003;102(1):276–83, http://dx.doi.org/10.1182/blood-2002-09-2896.

15. Quintas-Cardama A, Kantarjian H, Jones D, Nicaise C, O’Brien S, Giles F,et al. Dasatinib (BMS-354825) is active in Philadelphia chromosome-positive chronic myelogenous leukemia after imatinib and nilotinib(AMN107) therapy failure. Blood. 2007;109(2):497–9, http://dx.doi.org/10.1182/blood-2006-07-035493.

16. Giles FJ, Abruzzese E, Rosti G, Kim D-W, Bhatia R, Bosly A, et al. Nilo-tinib is active in chronic and accelerated phase chronic myeloid leukemiafollowing failure of imatinib and dasatinib therapy. Leukemia. 2010;24(7):1299–301, http://dx.doi.org/10.1038/leu.2010.110.

17. Marin D, Ibrahim AR, Lucas C, Gerrard G, Wang L, Szydlo RM, et al.Assessment of BCR-ABL1 transcript levels at 3 months is the onlyrequirement for predicting outcome for patients with chronic myeloidleukemia treated with tyrosine kinase inhibitors. J Clin Oncol. 2012;30(3):232–8, http://dx.doi.org/10.1200/JCO.2011.38.6565.

18. Hughes TP, Hochhaus A, Branford S, Müller MC, Kaeda JS, Foroni L, et al.Long-term prognostic significance of early molecular response to imatinibin newly diagnosed chronic myeloid leukemia: an analysis from theInternational Randomized Study of Interferon and STI571 (IRIS). Blood.2010;116(19):3758–65, http://dx.doi.org/10.1182/blood-2010-03-273979.

19. Hanfstein B, Müller MC, Hehlmann R, Erben P, Lauseker M, Fabarius A,et al. Early molecular and cytogenetic response is predictive for long-termprogression-free and overall survival in chronic myeloid leukemia (CML).Leukemia. 2012;26(9):2096–102, http://dx.doi.org/10.1038/leu.2012.85.

20. Branford S, Kim D-W, Soverini S, Haque A, Shou Y, Woodman RC, et al.Initial molecular response at 3 months may predict both response andevent-free survival at 24 months in imatinib-resistant or -intolerantpatients with Philadelphia chromosome-positive chronic myeloid leuke-mia in chronic phase treated with nilotinib. J Clin Oncol. 2012;30(35):4323–9, http://dx.doi.org/10.1200/JCO.2011.40.5217.

21. Shah NP, Guilhot F, Cortes JE, Schiffer C a, Le Coutre P, Brümmendorf TH,et al. Long-term outcome with dasatinib after imatinib failure in chronic-phase chronic myeloid leukemia: follow-up of phase 3 study. Blood.2014;123(15):2317-24, http://dx.doi.org/10.1182/blood-2013-10-532341.

22. Quintás-Cardama A, Kantarjian H, Jones D, Shan J, Borthakur G, ThomasD, et al. Delayed achievement of cytogenetic and molecular response isassociated with increased risk of progression among patients with chronicmyeloid leukemia in early chronic phase receiving high-dose or standard-dose imatinib therapy. Blood. 2009;113(25):6315–21, http://dx.doi.org/10.1182/blood-2008-07-166694.

23. Ibrahim AR, Paliompeis C, Bua M, Milojkovic D, Szydlo R, Khorashad JS,et al. Efficacy of tyrosine kinase inhibitors (TKIs) as third-line therapy inpatients with chronic myeloid leukemia in chronic phase who have failed2 prior lines of TKI therapy. Blood. 2010;116(25):5497–500, http://dx.doi.org/10.1182/blood-2010-06-291922.

24. Garg RJ, Kantarjian H, O’Brien S, Quintás-Cardama A, Faderl S,Estrov Z, et al. The use of nilotinib or dasatinib after failure to 2 priortyrosine kinase inhibitors: long-term follow-up. Blood. 2009;114(20):4361–8, http://dx.doi.org/10.1182/blood-2009-05-221531.

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http://dx.doi.org/10.5581/1516-8484.20110056


















The effect of elemene on lung adenocarcinoma A549cell radiosensitivity and elucidation of its mechanismKun Zou, Caigang Liu, Zhuo Zhang*, Lijuan Zou*

2nd Affiliated Hospital of Dalian Medical University, Radiotherapy Department, Dalian/Liaoning, China.

OBJECTIVE: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecularmechanism.

METHODS: Apoptosis of A549 cells was detected by flow cytometry and fluorescence microscopy. The effect ofdouble-strand break (DSB) damage repair in A549 cells was evaluated using the neutral comet assay. Proteinexpression levels were detected using western blotting, and the correlation between protein levels wasanalyzed.

RESULTS: Elemene exhibited a radiosensitizing effect on A549 cells. The level of apoptosis induced by elemenecombined with radiation was significantly greater (po0.01) than that elicited by either radiation or elemenealone. Following radiation and subsequent repair for 24 h, the tail intensity of A549 cells treated with acombination of elemene and radiation was greater than that of cells treated with either elemene or radiationalone (po0.01). This result indicates that elemene inhibits cellular DSB repair. Both elemene combined withradiation and radiation alone decreased the protein expression of DNA-PKcs and Bcl-2 compared to elemenealone (po0.01), while p53 protein expression was increased (po0.01). A negative correlation was observedbetween DNA-PKcs and p53 expression (r=-0.569, p=0.040), while a positive correlation was found betweenDNA-PKcs and Bcl-2 expression (r=0.755, p=0.012).

CONCLUSIONS: Elemene exhibits a radiosensitizing effect on A549 cells, and its underlying molecularmechanism of action may be related to the downregulation of DNA-PKcs gene expression.

KEYWORDS: Elemene; Radiosensitivity; A549 cells; DNA-PKcs; Bcl-2; p53.

Zou K, Liu C, Zhang Z, Zou L. The effect of elemene on lung adenocarcinoma A549 cell radiosensitivity and elucidation of its mechanism.Clinics. 2015;70(8):556-562

Received for publication on January 6, 2015; First review completed on February 27, 2015; Accepted for publication June 1, 2015

E-mail: [email protected], [email protected]

*Corresponding authors

’ INTRODUCTION

Lung cancer is one of the most common malignant tumors,and radiotherapy is the prevailing method of choice fortreatment (1), especially for middle- to late-stage lung cancer.Radiation works by damaging the DNA of cancerous cellsand altering apoptosis-related genes or proteins, leading tocell death. Improving the radiosensitivity of tumor cells is asignificant factor that would improve the efficacy ofradiotherapy.DNA-dependent protein kinase (DNA-PK) is an important

enzyme that participates in DNA damage repair and hasbecome the main target of radiation sensitivity interventions(2-4). The catalytic subunit (cs) of DNA-PK affects cellularradiosensitivity by regulating the phosphorylation of DNA

damage repair-related proteins (5). Thus, inhibition of DNA-PKcs gene expression can block DNA double-strand break(DSB) repair and improve cellular radiosensitivity.

Cellular apoptosis is the core characteristic of radio-therapy, and regulation of this process thus plays animportant role in cellular radiosensitivity (6,7). Previousstudies have shown that apoptosis-related genes, such asphosphoprotein (p53), p16, B-cell lymphoma-2 (Bcl-2), anderythroblastic leukemia viral oncogene homolog 2 (erbB-2),are associated with tumor radiosensitivity (8,9), especiallyp53 and Bcl-2. It has also been reported that elemeneinteracts with the frontier orbitals of DNA bases to formcomplexes between DNA molecules. Specifically, Jiang et al.(10) showed that elemene increases the radiosensitivity ofA549 cells, the mechanism for which may be related to theupregulation of p53, downregulation of Bcl-2, and inductionof cellular apoptosis.

Elemene, which is extracted from Zingiberaceae plants(Curcuma aromatica Salisb.), is a non-cytotoxic antitumorcompound that can improve the radiosensitivity of tumorcells (11). Results of an in vitro study showed that elemeneincreased the radiosensitivity of renal carcinoma cells,tongue squamous cancer cells, and non-small cell lungDOI: 10.6061/clinics/2015(08)05



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cancer cells (10,12,13). Animal experiments further showedthat elemene exhibited radiotherapy-sensitizing effects inmany types of tumor cells, such as transplanted murine U14tumors, kidney cancer GRC-1 cells, and tongue squamouscarcinoma Tca-8113 cells (13-15). In addition, beta elemeneenhances A549 cell radiosensitivity through enhancement ofDNA damage and suppression of DNA repair (16).In the current study, A549 cells were irradiated following

elemene treatment, and the changes in expression of theapoptosis-related genes Bcl-2 and p53 as well as the double-stranded DNA damage repair-related gene DNA-PKcs wereevaluated. These experiments were conducted to furtherunderstand elemene’s molecular mechanism of action inenhancing radiation sensitivity of A549 cells.


Cell cultureThe human lung adenocarcinoma A549 cell line was

purchased from the Chinese Academy of Medical Sciences(CAMS) Cell Center and passaged at the Second AffiliatedHospital of Dalian Medical University Center Laboratory.The cells were cultured in RPMI 1640 medium containing10% inactivated fetal bovine serum (FBS) at 37 ˚C under anatmosphere of 5% CO2 and saturated humidity. The cellswere subcultured when they reached the exponential phase.

Reagents and instrumentsElemene (0.1 g/20 mL), which was obtained from DaLian

JinGang Pharmaceutical Co. Ltd. (China), was dissolved inRPMI 1640 medium to final working concentrations of 10and 20 mg/mL before use. RPMI 1640 medium was obtainedfrom Gibco (USA); FBS was obtained from TianJin TBDBiotechnology Company (China); p53 and Bcl-2 antibodieswere obtained from Santa Cruz (USA, 1:1,000); and DNA-PKcs antibody (1:2,000), anti-human b-actin mouse mono-clonal antibody and histone H1 internuclear internalreference antibodies (1:200) were obtained from Neomarker(USA). The Jim-X half-dry transfer electrophoresis apparatuswas obtained from DaLian JingMai Biotechnology Co. Ltd.(China). The flow cytometer was purchased from the GeneCompany (USA). The CK2 type inverted microscope wasobtained from Olympus (Japan). The BX51 type fluorescentmicroscope was also obtained from Olympus (Japan).

Irradiation conditionsCell irradiation was performed using the Varian 2300C/D

medical linear accelerator (Varian Companies, USA) with acoverage field of 20 cm � 20 cm. The culture dish wasplaced in the radiation field above 1.5 cm of organic glass.Cells with irradiated with 6 MV X-ray irradiation at a dosagerate of 300 cGy/min, a rack angle of 180 ˚ , and source-to-surface distance (SSD) of 100 cm.

Clonogenic assayLogarithmic-growth phase cells were inoculated in a

60-mm culture dish. After adherence, the cells in the drugand combined irradiation groups were cultured in thepresence of 10 or 20 mg/mL elemene and seeded in cultureplates at 100 cells/well for 24 h. The cells were exposed to 0,2, 4, 6, 8, and 10 Gy of irradiation and cultured for another 14days. The number of cell clones viewed under a lowmagnification microscope was 50. The plating efficiency(PE) was calculated relative to the control group (0 Gy), and

the survival fraction (SF) of each group was calculated asfollows:

SER=control group (D0, Dq)/experimental group (D0, Dq).

Morphological assessment of apoptosisThe cells were treated as follows: the control group

received RPMI 1640 medium; the radiation group receiveda radiation dose of 4 Gy; the drug group was treated with 10or 20 mg/mL elemene; and the drug plus radiation groupwas treated with 10 or 20 mg/mL elemene followed by aradiation dose of 4 Gy. Following incubation with elemene,the exponentially growing cells were irradiated as describedabove. Samples of 3 � 105 cells were then collected fromeach group, treated with pancreatic enzyme digesting cells,rinsed twice with PBS, and centrifuged at 1,000 rpm for5 min. The SF was determined using the following equation:

SF=colony number/(plating cell number � PE).

The dose survival curve was fitted using the linear-quadratic (LQ) function model S=e-(ad+bd2) (17) for calculat-ing radiobiological parameters, including the sensitivityenhancement ratio (SER), SER of the mean lethal dose (D0)SERDq, and SER of the quasi-threshold dose (Dq) SERD0

.Nuclear morphology was examined using fluorescencemicroscopy following Hoechst 33342 staining (final concen-tration 8 mg/mL) for 15 min at 37 ˚C. Imaging wasperformed using an Olympus BX-51 fluorescent microscopewith appropriate filter cubes. The excitation and emissionwavelengths were 350 nm 460 nm, respectively.

Apoptosis standardNormal cells demonstrated uniform dispersion of low-

density fluorescence, while apoptotic cells showed high-density fluorescence, characterized by a bright blue hue.

Assessment of apoptosisThe cells used were grouped and treated as specified

above. Then, samples of 3 � 105 cells were collected fromeach group, treated with pancreatic enzyme digesting cells,rinsed twice with PBS, and centrifuged at 1,000 rpm for5 min. The cells were treated with 100 mL 2% Triton X-100 for20 min, rinsed twice with PBS, and centrifuged at 1,000 rpmfor 5 min. Next, 200 mL of DNA-Prep LPR reagent (Beckman-Coulter Ltd) was added for 20 min, and the cells were rinsedtwice with PBS, followed by centrifugation at 1,000 rpm for5 min. The cells were resuspended in PBS and 50 mg/mLpropidium iodide (PI) reagent containing 480 mL of PBS, 5 mLof PI (5 mg /mL), and 5 mL of RNase (10 mg /mL), and10 mL of Triton X-100 (10%) was added 30 s later. Single-cellsuspensions were analyzed by flow cytometry to determinethe cellular apoptosis rate.

Neutral comet assayA549 cells were irradiated in the absence or presence of

elemene (10 or 20 mg/mL) and assayed immediately afterradiation or returned to the incubator for 24 h to permitrepair. The comet assays were performed immediately afterincubation with elemene, irradiation or combination treat-ment. Tumor cells were grown on glass microscope slidesusing a standard protocol (18). The slides were submerged inlysing solution containing 30 mM ethylenediaminetetraacetic

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CLINICS 2015;70(8):556-562 Effect of elemene on lung cancerZou K et al.

acid (EDTA) and 0.5% sodium dodecyl sulfate (SDS, pH 8.3)for 1.5 h at 37 ˚C. Following lysis, the slides were rinsed threetimes in Tris-borate-EDTA (TBE) buffer consisting of 90 mMTris, 90 mM boric acid, and 2 mM EDTA, pH 8.5, and storedovernight in TBE buffer at 4 ˚C. Slides were transferred to anelectrophoresis unit with TBE buffer and electrophoresed at1 V/cm for 20 min. Following electrophoresis, the slideswere neutralized with 0.4 M Tris buffer (pH 7.5) and stainedwith ethidium bromide (20 mg/mL). Finally, the slides wereviewed using an Olympus BX-51 fluorescent microscope(excitation filter 549 nm, barrier filter 590 nm). Images of 50randomly selected cells from each slide were analyzed withComet Assay Software Project casp-1.2.2 (University ofWroclaw, Poland). The tail moment was used as a parameterto assess DNA damage. The assay was completed threeseparate times, and 50 cells were evaluated per experiment.

Western blot assayWestern blotting was performed to detect the expression

levels of DNA-PKcs, p53, and Bcl-2. The cells were groupedand treated as specified above. After 24 h, western blotanalysis was performed using cytosolic fractions as pre-viously described (19). Equal amounts of cytosolic proteinwere separated on 8–12% SDS polyacrylamide denaturinggels and transferred to nitrocellulose membranes. Themembranes were blocked in TBS-Tween (TBS-T, 10 mMTris-HCl, pH 7.4; 150 mM NaCl; and 0.1% Tween-20) with5% non-fat milk for 2 h and incubated with specific primaryantibodies overnight at 4 ˚C. Finally, the membranes wereincubated with horseradish peroxidase (HRP)-conjugatedsecondary antibodies at 37 ˚C for 2 h and assayed using anenhanced chemiluminescence plus detection system.

Statistical analysisData were analyzed using the statistical package for the

social sciences (SPSS) v13.0 software. Data are expressed asthe mean ± standard deviation (SD). The statisticalsignificance of differences between groups was determinedby one-way analysis of variance (ANOVA), followed by posthoc analysis using the least significant difference (LSD) formultiple comparisons. The Spearman test was used for thecorrelation analysis of the relationships between the expres-sion levels of genes. The level of significance was set atpo0.05 and po0.01 for all statistical analysis.

’ RESULTS

Effects of elemene on cell radiosensitivityThe survival fraction of A549 cells decreased following

treatment with different doses of radiation and the sameconcentration of elemene. Conversely, the A549 cellsurvival fraction decreased in the groups treated with thesame dose of radiation in combination with increasingconcentrations of elemene (Table 1). Following treatmentwith 10 or 20 mg/mL elemene, the A549 cell survival curveshifted to the left, the shoulder area was diminished, andthe steepness of the curve increased (Figure 1). Based onthe cell survival curve, the radiobiological parameters andradiosensitization ratio were obtained and are listed inTable 1. These data show that, compared with the controlgroup, the SERD0

and SERDq values for the 10 and 20 mg/mLelemene treatment groups were greater than 1. Furthermore,the ratio gradually increased with an increasing drugconcentration (Table 2).

Effect of elemene on A549 cell apoptosisFluorescence microscopy showed that compared with the

control group, the groups treated with radiation alone andelemene alone contained more apoptotic cells (Figure 2).Furthermore, significantly higher apoptotic levels wereobserved in the groups treated with radiation and elemeneat 10 or 20 mg/mL (Figure 2A). Flow cytometry revealed that

Table 1 - Survival fraction (%).

Group 0 Gy 2 Gy 4 Gy 6 Gy 8 Gy 10 Gy

Control 100 84.1±13.2 70.2±10.5 66.3±1.9 47.3±2.5 26.0±1.310 mg/ml 97.7±20.2 76.2±10.4 49.5±6.4 22.5±2.3 7.5±1.8 3.4±0.220 mg/ml 95.4±18.8 63.6±7.5 30.4±3.0 5.7±1.1 2.7±0.6 0.7±0.1

Cells were incubated with 10 or 20 mg/mL concentrations of elemene for 24 h and were then irradiated with 0, 2, 4, 6, 8, and 10 Gy and cultured foranother 14 days. The number of cells forming more than 50 clones was counted under the inverted microscope to calculate the cloning efficiency (CE):CE (%)=(Clone formation average of treatment group/inoculated cell number) � 100%. Survival fraction (SF)=(irritated group CE/non-irradiated groupPE) � 100%. The experiment was repeated 3 times to calculate the average.

Figure 1 - Cell survival curves of A549 cells following treatmentwith 0, 10 or 20 mg/mL elemene and exposure to x-ray doses from0 to 10 Gy.

Table 2 - Radiation parameters for a single-hit multi-targetmodel.

Group D0 (Gy) Dq (Gy) SF2 (%) SERD0SERDq

Control 2.54±0.24 2.68±0.25 84.6±20.9 - -10 mg/mL 1.64±0.15 1.87±0.22 56.3±14.9 1.54±0.20 1.43±0.1520 mg/mL 1.55±0.13 1.53±0.11 43.2±10.7 1.63±0.32 1.75±0.19

SPSS17.0 was used to calculate survival parameters including SF2(surviving fraction of 2 Gy), D0 (mean lethal dose or final slope), andDq (quasi-thres-hold dose). SER (sensitization enhancement ratio). SER D0

(Dq)=control group D0 (Dq)/drug group D0 (Dq) . Three parallel sampleswere set at each radiation dosage.

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CLINICS 2015;70(8):556-562

Figure 2 - Elemene induces apoptosis in A549 cells. Cells were incubated with 10 or 20 mg/mL elemene for 24 h, followed by irradiation with4 Gy X-rays and washing with PBS. (A) Cells were imaged with an Olympus BX-51 fluorescent microscope using appropriate filter cubes. (B)Chromatin condensation was analyzed by fluorescence microscopy after DNA staining with Hoechst 3334. The results are expressed as themean ± SD of three independent experiments, n=3, **po0.01 compared to control, ##po0.01 compared to radiation alone.

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compared with the control group, the apoptosis rate of thegroup treated with radiation alone appeared to increase, butthis was not found to be statistically significant (p40.05). Theapoptosis rate of the group treated with elemene alone didnot change (p40.05), while the apoptosis rate of the elemeneplus radiation group increased significantly (po0.01). Therate of apoptosis increased with increasing concentrations ofelemene (Figure 2B).

Effect of elemene on DSB repair in A549 cellsFigure 3 (0 h) shows that both elemene and irradiation

alone increased the tail intensity in A549 cells, and thedegree of DSB was augmented as the elemene concentrationincreased. In particular, the results showed a higher numberof DSB in the combination group than the irradiation aloneand elemene alone groups (po0.01). As shown in Figure 3(24 h), following incubation for 24 h, the tail intensity in theirradiation alone group returned to background levels,while there was a significant increase in the amount ofremaining tail intensity in the combination group comparedwith the irradiation alone and elemene alone groups(po0.01).

Effects of elemene on DNA-PKcs, Bcl-2, and p53protein expression in A549 cellsThe results shown in Figure 4A revealed that in the 10 and

20 mg/mL combined treatment groups, a significant decrease inthe protein expression of DNA-PKcs (po0.01, Figure 4B) andBcl-2 (po0.01, Figure 4C) was observed, while the p53 proteinexpression level was significantly increased (po0.01, Figure 4C).

Protein expression correlation analysisSpearman correlation analysis showed that DNA-PKcs

and p53 protein expression was negatively correlated(r=-0.569, po0.05), while DNA-PKcs expression was posi-tively correlated with Bcl-2 protein expression (r=0.755,po0.05).

’ DISCUSSION

Basic research in radiation biology has shown thatradiation therapy works mainly by damaging tumor cellDNA and altering the expression of apoptosis-related genesand proteins. The radiosensitivity of tumor cells relates totheir capacity to repair DSB via the related genes DNA-PKcs, Ku70/80, and ataxia telangiectasia mutated (ATM).Other genes known to be involved in radiosensitivity andresponsible for apoptosis regulation include p53, Bcl-2,c-myc proto-oncogene (c-myc), and survivin (9). Betaelemene, which is the active component of elemene, hasrecently been demonstrated to enhance the radiosensitivityof human cancer cell lines in vitro and in an animal tumormodel in vivo (16,20). In particular, beta elemene was foundto enhance radiosensitivity by influencing the cell cycledistribution of gastric cancer MKN28 cells, and themechanisms responsible for this effect include the inductionof G2/M phase arrest, inhibition of sublethal damage repair,and induction of cell apoptosis, which enhances the killingeffects of radioactive rays (21). The results of the currentstudy show that the SERD0

and SERDq values of A549 cellsexposed to a low concentration of cytotoxic elemene weregreater than 1. In addition, elemene enhanced the sensitivity

Figure 3 - Influence of elemene on radiation-induced DSB. A549 cells were irradiated with 4 Gy X-rays in the absence or presence ofelemene (10 or 20 mg/mL) and assayed immediately after radiation or returned to the incubator for 24 h to permit repair. Comet assayswere performed immediately after incubation with elemene treatment, irradiation, or combination treatment. Columns represent themeans of the tail moments from three independent experiments, and the bars represent the SD, **po0.01 vs. control, ##po0.01compared to radiation alone.

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of A549 cells to radiotherapy. Cellular apoptosis is funda-mental to radiotherapy, and its regulatory mechanism playsan important role in cellular radiosensitivity. Apoptosis-related genes such as p53 and Bcl-2 have importantregulatory functions in the progression of rapid apoptosisinduced by radiation therapy. For instance, a previous studyshowed that the levels of the antiapoptotic genes Bcl-2 andBcl-xl in A549 cells decreased, while p53 expression and theproduction of exosomes increased, following elemenetreatment (22). This result demonstrates that both p53 andBcl-2 have important regulatory actions in cervical cancercell apoptosis induced by radiation. A number of experi-mental studies have further shown that elemene is involvedin regulating the expression of Bax, c-myc, p53, poly (ADP-ribose) polymerase (PARP), survivin, and livin as well asinducing tumor cell apoptosis (23-26). Our results showedthat, compared with the exposure alone group, the groupthat received elemene combined with irradiation exhibitedincreased p53 gene expression and significantly decreasedBcl-2 gene expression, and the expression of both genes wassignificantly correlated. Furthermore, elemene was shown toregulate expression of the apoptosis-related genes Bcl-2 and

p53 and induce A549 cell apoptosis, thereby increasing cellradiosensitivity.Interestingly, when Bcl-2 and p53 gene expression was

significantly altered, DNA-PKcs protein expression wassignificantly decreased in the combined treatment group.This result indicates that elemene is also involved inregulating DNA damage repair pathways. Protein kinaseactivation leads to the phosphorylation of downstreamDNA repair proteins, which initiate DNA chain fracturerepair (27), and the relationship between DNA-PKcs andradiotherapy sensitivity has been a topic of significantresearch in recent years. It is well established thatinhibiting tumor cell expression of DNA-PKcs increasesradiation sensitivity. Panet al. (28) studied the relationshipbetween DNA-PKcs expression and radiation sensitivity innon-small cell lung cancer cell lines, and in adenocarcino-mas and large cell carcinomas, DNA-PKcs was shown tobe an important component regulating cellular radio-sensitivity. This result indicates that DNA-PKcs may bepredictive of non-small cell lung cancer cell radiosensitiv-ity. Zou et al. (29) silenced the DNA-PKcs gene of humanmammary epithelial cells (MCF10F) using small interfer-ing RNA (siRNA) technology. Simultaneously, the expres-sion of DNA repair-related proteins, such as DNA-PKcs,Ku80, ATM, and p53, was decreased in these cells, whiletheir sensitivity increased with low doses of radiation.Small molecule inhibitors of DNA-PKcs were also shownto enhance radiation sensitivity of cervical cancercells (30). Our experimental results showed that elemeneinhibited DNA-PKcs expression in A549 cells,reduced DNA damage repair, and increased cellularradiosensitivity.DNA-PKcs is a protein with a wide range of functions and

is involved in DNA damage repair, apoptosis, and V(D)Jrecombination (31). Yu et al. (32) found that in non-small celllung cancer, high expression of DNA-PKcs increased theactivity of the DNA damage repair system. In addition,apoptosis inhibition caused by mutant p53 and Bcl-2expression exhibited a combined effect, which may explainthe development of resistance to radiotherapy in small-celllung cancer. Daido et al. (33) indicated that followingexposure to low doses of radiation, human malignant gliomaM059J cells that lack DNA-PKcs underwent massive autop-hagic cell death that was significantly increased afterexposure to DNA-PKcs inhibitors. Furthermore, DNA-PKcsinhibitors exert radiotherapy-sensitizing effects on gliomacells by enhancing type II programmed cell death. Lee et al.(34) found that p53-inducible gene 3 (PIG3) is involved inapoptosis caused by p53 activation and that this moleculecan regulate DNA-PKcs expression. Moreover, knockdownof PIG3 was shown to decrease the level of DNA-PKcs incells. Our study further addressed the correlation betweenDNA-PKcs, Bcl-2, and p53 expression, and the resultsshowed that DNA-PKcs expression was significantly posi-tively correlated with that of Bcl-2 (r=0.755, po0.05) andsignificantly negatively correlated with p53 (r=0.569,po0.05). We further showed that DNA-PKcs was closelyrelated to apoptosis and that elemene increased apoptosis ofA549 cells and strengthened cellular radiosensitivity byinhibiting DNA-PKcs expression.In summary, elemene exhibits radiotherapy-sensitizing

effects on lung adenocarcinoma A549 cells, and its mechan-ism of action involves the upregulation of p53 and down-

Figure 4 - Western blot analysis of the protein levels of Bcl-2, p53and DNA-PKcs. A549 cells were irradiated with 4 Gy X-raysfollowing treatment with 10 or 20 mg/mL elemene for 24 h.Proteins were extracted and separated by SDS-PAGE. (A) Levels ofBcl-2, p53 and DNA-PKcs were quantitated by densitometry, andthe ratios of the three proteins are displayed. Values representthe mean ± SD, n=3, **po0.01 compared to the control,##po0.01 compared to radiation alone. Elemene inhibited theprotein expression of (B) DNA-PKcs and (C) Bcl-2 and promotedthe protein expression of p53. Key: 1, Control; 2, irradiation; 3,10 mg/mL; 4, 20 mg/mL; 5, 10 mg/mL + irradiation; 6, 20 mg/mL +irradiation.

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regulation of Bcl-2 to promote cell apoptosis, as well as thedownregulation of DNA-PKcs to inhibit DSB repair. How-ever, the specific mechanism of action of elemene requiresfurther elucidation.

’ ACKNOWLEDGEMENT

This study was supported by grants from the National Natural ScienceFoundation of China (No. 81473452).


Zou K, Zhang Z and Zou L designed the research study and wrote thepaper. Zou K and Liu C performed the research. Zou K and Zhang Zanalyzed the data.

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http://dx.doi.org/10.1016/j.rpor.2014.04.012

http://dx.doi.org/10.1080/09553000210166606

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http://dx.doi.org/10.3109/07357909909011718

http://dx.doi.org/10.1007/s10330-008-0139-3

http://dx.doi.org/10.1007/s13277-014-2226-0

http://dx.doi.org/10.3736/jcim20060415

http://dx.doi.org/10.1002/ptr.v25.7

http://dx.doi.org/10.3109/0284186X.2014.925582

http://dx.doi.org/10.3109/0284186X.2014.925582

http://dx.doi.org/10.1093/mutage/gep048


http://dx.doi.org/10.1089/cbr.2011.1003

http://dx.doi.org/10.1016/j.lungcan.2014.08.015

http://dx.doi.org/10.1016/S0022-2836(02)01328-1

http://dx.doi.org/10.1158/0008-5472.CAN-04-4202

http://dx.doi.org/10.1038/onc.2009.438

Non-alcoholic steatohepatitis-related liver cirrhosis isincreasing in China: A ten-year retrospective studyJi Xiong,I Jun Wang,II Juan Huang,I Wenjing Sun,I Jun Wang,I Dongfeng ChenI,*

IThird Military Medical University, Daping Hospital, Institute of Surgery Research, Department of Gastroenterology, Chongqing, China. IIMedical Team of

Chinese People’s Armed Police Force, Xinjiang, China.

OBJECTIVE: Little is known about metabolic factors in cirrhotic patients in China. Therefore, we aimed to quantifythe prevalence of both metabolic factors and non-alcoholic steatohepatitis-related liver cirrhosis in China.

METHODS: The medical records of 1,582 patients diagnosed with liver cirrhosis from June 2003 to July 2013 atDaping Hospital (Chongqing, China) were retrospectively reviewed through a computer-generated search.

RESULTS: Serum hepatitis B virus surface antigen was present in 1,083 (68.5%) patients, and hepatitis B was foundto be the only etiological factor in 938 (59.3%) of all patients. Obesity, diabetes mellitus, and arterial hypertensionwere observed in 229 (14.5%), 159 (10.1%), and 129 (8.2%) patients, respectively. From 2012-2013, the proportionof non-alcoholic steatohepatitis-related liver cirrhosis increased to 3.2%, whereas the average proportion of non-alcoholic steatohepatitis-related liver cirrhosis in the previous ten years was 1.9%. The incidence of hepatocellularcarcinoma was much higher in males than in females (6.3% vs. 3.7%, respectively, p=0.036). Obesity and diabetesmellitus did not significantly increase the incidence of hepatocellular carcinoma in the whole cirrhotic group. Thepresence of hepatitis B virus was the only risk factor for hepatocellular carcinoma in cirrhotic patients (po0.001).

CONCLUSIONS: Although hepatitis B virus remains the main etiology of liver cirrhosis in China, steatohepatitis-related liver cirrhosis is increasing in frequency. Hepatitis B virus was the sole significant risk factor forhepatocellular carcinoma in the whole cirrhotic group in the present study, in contrast to obesity and diabetesmellitus, for which only a trend of increased hepatocellular carcinoma was found.

KEYWORDS: Non-Alcoholic Steatohepatitis; Non-Alcoholic Fatty Liver Disease; Liver Cirrhosis; Obesity; Metabolism.

Xiong J, Wang J, Huang J, Sun W, Wang J, Chen D. Non-alcoholic steatohepatitis-related liver cirrhosis is increasing in China: A ten-yearretrospective study. Clinics. 2015;70(8):563-568

Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on June 1, 2015



’ INTRODUCTION

The main etiology of liver cirrhosis (LC) in China ishepatitis B virus (HBV) infection (1). However, the changingproportions of the various potential etiological factors overthe last 10 years remain poorly documented, especially thepresence of metabolic characteristics such as obesity (orincreased body mass index [BMI]), diabetes mellitus (DM),hypertension and alcoholism. In Western countries, non-alcoholic steatohepatitis (NASH)-related cirrhosis is increas-ingly considered as a major causal factor of cirrhosis.Moreover, hepatocellular carcinoma (HCC) is related toNASH-related cirrhosis in Germany (2). For several possiblereasons, few data exist regarding NASH-related LC in China.First, NASH-related cirrhosis is diagnosed by exclusion, andruling out all other possible causes is difficult. Second, the

histological characteristic features of non-alcoholic fatty liverdisease (NAFLD) such as fatty infiltration tend to be lost inNASH-related cirrhosis (3). Third, there is a lack of strictcriteria for diagnosing NASH-related cirrhosis. According toone nationwide Japanese survey, 2.1% of cirrhosis cases wereattributable to NASH-related cirrhosis based on the research-ers’ own criteria, which mainly diagnosed NASH-relatedcirrhosis as cryptogenic cirrhosis associated with obesity(BMI over 25), DM or metabolic syndrome (Met-s) (4).If appropriate criteria are used, NASH-related cirrhosis

may account for two thirds of cases currently labeled ascryptogenic cirrhosis (3). Herein, we retrospectively analyzedetiological factors in LC in patients admitted to our hospitalover the past 10 years to evaluate the proportion and timetrend of NASH-related LC, with special emphasis on thefactors favoring HCC development.

’ PATIENTS AND METHODS

The medical records of patients diagnosed with LC fromJune 2003 to July 2013 at Daping Hospital (Chongqing,China) were retrospectively reviewed through a computer-generated search. The study included 1,582 patients.DOI: 10.6061/clinics/2015(08)06


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LC was diagnosed based on clinical symptoms, imagingdata, and/or histological findings (only 41 patients had aliver biopsy in the present series). Classical criteria for eachetiology potentially involved in LC were applied tocategorize cirrhotic patients. For NASH-related LC, weadopted the following criteria. First, all NASH-related LChad to be cryptogenic LC and had to be coupled with alcoholconsumption of less than 20 grams per day. Second, the BMIof each patient had to be over 25, with or without DM orMet-s; this was slightly different from the Japanese criteria(4), which did not include obesity as a necessary criterion. Ifthe above two criteria were met, then the patient wasclinically suspected of having NASH-related LC. Further-more, if histological findings such as micronodular cirrhosis,perisinusoidal fibrosis or fatty changes were found, then thepatient was histologically diagnosed with NASH-related LC.Among the 30 patients diagnosed with NASH-related LC,only one patient was histologically diagnosed with NASH-related LC by liver biopsy.Clinical information (age, gender, alcohol consumption,

virus infection, hepatic complications) was also collected forall patients with LC. Metabolic risk factors such as obesity,DM and arterial hypertension were systematically collectedbased on the diagnostic history. The Child-Pugh criteria wereused to grade cirrhosis.

Statistical analysisThe dichotomous data and continuous data were analyzed

by the chi-square test and Student’s t test, respectively, usingStatistical Package for the Social Sciences (SPSS, version 15.0;Chicago, IL, USA). Logistic regression was used to analyzethe selected risk factors for HCC. When examining thedifferences between males and females, Fisher’s exact testand the Mann-Whitney U test were also used. A p-value ofo0.05 was considered statistically significant.

’ RESULTS

Etiology of LCA total of 1,582 patients diagnosed with LC from 2003-

2013 were included in this retrospective study. A total of1,097 (69.4%) patients were male, 485 (30.6%) were female,and 87 (5.5%) were diagnosed with HCC duringhospitalization.In our study, 14 etiological categories were set for LC

(Table 1). The most common etiology was HBV; hepatitis Bsurface antigen (HBs Ag) was present in the sera of 1,083(68.5%) patients. HBV was found to be the only etiologicalfactor in 938 (59.3%) patients. Hepatitis C virus (HCV)accounted only for 1.8% of cases (1.0% of patients had HCValone, 0.5% had HCV combined with HBV, and 0.3% hadHCV combined with alcohol abuse). Hepatitis E viruscombined with HBV was found in only two patients. Intotal, 298 (18.7%) patients had a history of alcohol abuse (135of them also had HBV infection, and 5 patients also had HCVinfection; alcohol was the dominant factor in 158 patients).There were 32 patients with autoimmune hepatitis (AIH), 69patients with primary biliary cirrhosis (PBC), 4 patientswith AIH combined with PBC, and 6 patients withWilson’s disease. For NASH-related LC, 30 patients (1.9%)met our criteria. Thirteen patients had other identi-fied etiologies, including intra-hepatic bile-duct stones(9 patients), Budd-Chiari syndrome (2 patients), parasiticinfection (1 patient), and veno-occlusive disease (1 patient).

The remaining 167 patients had cryptogenic LC, as defined bythe absence of identified causes, including NASH-related LCcriteria.

Etiological differences between males and femalesHBV was clearly the main etiology in both males and

females (59.8% vs. 57.9%, respectively, p40.05) (Figure 1).The percentages of patients with alcohol abuse, HBV plusalcohol abuse, and HCV plus alcohol abuse were higher inmales vs. females (11.2% vs. 1.0%, po0.01; 12.2% vs. 0.2%,po0.01; and 0.5% vs. 0%, po0.01, respectively). However,the percentages of AIH and PBC were much higher infemales vs. males (4.9% vs. 0.7%, po0.01; 12.6% vs. 0.7%,po0.01, respectively). No difference between males andfemales existed for NASH-related LC (1.7% vs. 2.3%,respectively, p=0.47).

Time trend of metabolic factors and HCC incirrhotic patients

The metabolic information recorded during the periodsfrom 2003-2008 and 2008-2013 was compared (Table 2).Whereas the total number of patients from 2008-2013 wastwo times higher than the number in the period from 2003-2008 (1,091 and 491, respectively), no significant differenceswere noted in terms of age, sex ratio, or BMI between the twotime periods. Trends of increased obesity, DM, hypertensionand HBs Ag positivity were observed in the second five-yearperiod. In contrast, the proportion of HCC significantlydecreased in the period from 2008-2013 (4.7% vs. 7.3%,p=0.033) (Table 2).

NASH-related LCAmong the total of 197 patients with cryptogenic LC, 30

patients met our criteria for NASH-related LC. As shown inFigure 2, the proportion of NASH-related LC increased inthe last two years of the study to 3.2%, compared with 1.9%for the whole study period.

Table 1 - Etiologies of liver cirrhosis from 2003-2013.

Category No. of patients (%)

HBV alone 938 (59.3)HCV alone 15 (1.0)HBV/HCV 8 (0.5)HBV/HEV 2 (0.1)Alcohol 158 (9.9)HBV/Alcohol 135 (8.5)HCV/Alcohol 5 (0.3)AIH 32 (2.0)PBC 69 (4.4)AIH/PBC 4 (0.3)Wilson’s disease 6 (0.4)NASH-LC 30 (1.9)Cryptogenic 167 (10.6)Other 13 (0.8)Total 1,582 (100)

HBV alone: hepatitis B virus was the only etiological factor for cirrhosis;HCV alone: hepatitis C virus was the only etiological factor for cirrhosis;HBV/HCV: hepatitis B virus combined with hepatitis C virus; HBV/HEV: hepa-titis B virus combined with hepatitis E virus; HBV/Alcohol: hepatitis B viruscombined with alcohol abuse; HCV/Alcohol: hepatitis C virus combinedwith alcohol abuse; AIH: autoimmune hepatitis; PBC: primary biliary cir-rhosis; AIH/PBC: autoimmune hepatitis combined with primary biliarycirrhosis.

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Among the 30 patients with NASH-related LC, 19 patientswere men, and 11 were women (no statistical significance).Ascites and upper gastrointestinal bleeding (UGB) were themain complications in patients with NASH-related LC, and thecomplication rate of both ascites and UGBwas higher in women,but the difference was not significant. For Child-Pugh grading ofNASH-related LC, the rate of Child Awas lower in women thanin men, but the difference was not significant. HCC was notfound in women or men with NASH-related LC. (Table 3).

Selected risk factors for HCCAmong the 87 patients with HCC, 69 patients were men,

and 18 patients were women. As shown in Table 4, only thesex ratio (male to female) and HBs Ag positivity rate weresignificantly increased in HCC patients compared with non-HCC patients (po0.05) by univariate analysis. Metabolicfactors, which included obesity, DM, and hypertension, werenot significantly different between men and women. HBs Agpositivity was the sole risk factor for HCC in cirrhoticpatients (po0.001) in a multivariate analysis. Obesity andDM did not increase the HCC incidence in the wholecirrhotic group (5.7% vs. 5.5%, respectively, p=0.862; 5.0% vs.5.6%, respectively, p=0.849). However, among patients withcirrhosis induced only by HBV, a trend of increased rates ofHCC in obese and DM patients was found (8.4% vs. 6.5%,respectively, p=0.436; 7.1% vs. 6.7%, respectively, p=0.589).

’ DISCUSSION

In our study, HBV remained the main etiological factor(68.5%) for LC in China, whereas HCV was by far the mostfrequent cause of cirrhosis (60.9%) according to a nationwidesurvey in Japan (4). HCV accounted for only 1.8% of ourcirrhotic patients. Alcoholic liver disease and hepatitis C arethe most common causes of cirrhosis in the Western world,whereas hepatitis B is the prevailing cause in most parts ofAsia and sub-Saharan Africa (5). For example, in the UnitedKingdom (6), a total of 3,360 incident cases of cirrhosis inpatients aged 25 or older were diagnosed with LC between1992 and 2001, and 35.9% of these cases were induced byalcohol, whereas only 5.4% were induced by viral hepatitis.In the USA, 7% of cirrhosis cases were attributable to HBV,and 27% were to HCV from 1989-2000(7). Globally, 57% ofcirrhosis cases were attributable to either HBV (30%) or HCV(27%) (7). In China, as reported in our study, 59.3% ofcirrhosis cases were only related to HBV infection. In a largecommunity-based study in eastern China (8), a total of149,175 individuals were investigated in 60 communities inthree counties in Jiangsu province from 2011-2012. Of thesesubjects, 1,175 (0.79%, 95% confidence interval (CI) 0.74-0.83)were anti-HCV antibody positive. The prevalence was evenlower in children (0.09%, 95% CI 0.04-0.17). A seroepide-miological survey of HCV conducted in 2007 revealed alower rate of anti-HCV antibody positivity (0.80%) inChongqing, Southwest China than in the general population(9). Thus, the prevalence of hepatitis C as a cause of LC is lowin Chongqing, China.The difference between Asia or sub-Saharan Africa and the

Western world may be related to the wide implementation ofHBV programs, which has not been financially possible innumerous developing countries. In 1992, the World HealthOrganization (WHO) recommended that all countriesinclude a hepatitis B vaccine in their routine infantimmunization programs. As of 2003, the WHO/UNICEFestimated 42% hepatitis B vaccination coverage in the globalbirth cohort (7). In China, the HBV infection prevalencedecreased from 9.7% (in 1992) to 7.2% (in 2006) after theintroduction of a universal HBV vaccination program in 1988(10). However, in our study, we did not observe thetheoretically expected decrease in the development of HBV-induced LC, possibly because HBV infection was present

Figure 1 - Difference in etiologies between males and females.

Table 2 - Clinical characteristics of cirrhotic patients.

Total 2003–2008 2008–2013 p-value#

Age (years) 52.8±12.8 52.3±12.8 52.7±12.7 0.519Female 485 147 338 0.678Male 1,097 344 753Male/female 2.26 2.34 2.23BMI (Mean ± SD) 22.1±3.3 22.1±3.5 22.1±3.2 0.894Obesity (%) 229 (14.5) 71 (14.5) 158 (14.5) 0.991DM (%) 159 (10.1) 44 (8.9) 115 (10.5) 0.334Hypertension (%) 129 (8.2) 33 (6.7) 96 (8.8) 0.162HBs Ag+ (%) 1,083 (68.5) 325 (66.2) 758 (69.5) 0.193HCC (%) 87 (5.5) 36 (7.3) 51 (4.7) 0.033Number of patients 1,582 491 1,091

BMI: body mass index; DM: diabetes mellitus; HBS Ag+: hepatitis B virussurface antigen positive; HCC: hepatocellular carcinoma.#p-value comparing 2003–2008 with 2008–2013.

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prior to the start of the vaccination campaign; the develop-ment of cirrhosis usually requires a long period of time (11).Nevertheless, the efficacy of the Chinese HBV immunizationprogram is indicated by the decreased prevalence of HBVcarrier status to 2.1% among all children and to 1.0% amongthose born after 1999, together with the presence of anti-HBsantibodies (1,12). Therefore, it is likely that HBV-relatedetiology of Chinese LC will be significantly reduced in thefuture.In Western countries, NAFLD is now considered to be the

most common cause of chronic liver disease, with 20-30% ofthe total population presenting with NAFLD (13,14). InChina, NAFLD affects approximately 15% of adults and ispresent in 68.5% of obese children (15, 16). The proportion ofNASH-related LC (1.9%) in our study was similar to that inJapan (2.1%) (4). However, our study was retrospective andmay not represent the incidence of LC in the generalpopulation. In our study, an increasing frequency was notedover the ten years covered by the study; this trend is likely toresult in a significant increase in patients with NASH-relatedLC in the future.A limitation of the current study, especially in the context

of a retrospective study, is that diagnosing LC related toNASH requires a BMI higher than 25. However, our criteriawere based on those adopted by the Japanese nationwide

study, with the difference that a BMI higher than 25 wasnecessary but not the sole criterion to consider cryptogeniccirrhosis as NASH-related cirrhosis because abnormalglucose metabolism, arterial hypertension and high trigly-ceride levels in the patients were also considered. AlthoughNASH can be found in lean patients, the identification ofmetabolic features or performance of a liver biopsy isrequired for an accurate diagnosis. Due to the retrospectivenature of our study, we could not document the number oflean patients; thus, we adopted a BMI higher than 25 as anecessary criterion. Notably, a liver biopsy may lack relevantmetabolic features when cirrhosis is present and thereforemay not always contribute to determining the etiology.

Although we considered obesity and DM in our studywhen diagnosing NASH-related LC, we did not findsignificant differences in the proportions of obesity, DMand hypertension when comparing the 2003-2008 and 2008-2013 time periods.

Several studies have reported that Met-s (obesity or DM)not only promotes the development of LC but also increasesthe risk of HCC (17-19). In China, obesity has become animportant health problem because the prevalence of obesityin adults, which was 7.1% in 2002, has increased by 97.2%since 1992 (20,21). In children, a dramatic increase in obesityhas been observed, with a two- to three-fold increase inBeijing and Shanghai between 1985 and 1995. In addition,the prevalence of overweight and obese children approached29% in 2000 in 7- to 12-year-old boys and 15-17% in girls (21).A 2013 study from Tianjing, China, also found that theprevalence of obesity in children under 7 years oldapproached 8.2% (22). These data illustrate the rapiddevelopment of obesity in both adults and children. In theUSA, data from the 2009-2010 National Health and NutritionExamination Survey reveal that 36% of Americans are obeseand that 30% of the general adult population may haveNAFLD (23). In the USA, the yearly cumulative HCCincidence in NASH-related LC was 2.6%, compared with4.0% among patients with HCV-related cirrhosis, over anaverage follow-up period of 3.2 years (24). Therefore, therapid development of obesity in China could be contributingto an increase in NAFLD, leading to the increased prevalenceof NASH-related LC shown in our study. The importance ofobesity in the natural history of NASH-related LC explains

Figure 2 - Time trend of NASH-related LC over the ten year periodfrom 2003-2013.

Table 3 - Clinical characteristics of NASH-related LC.

Total Female (F) Male (M) p-value (F vs. M)

Number of patients 30 11 19Age (years) 57.7±14.1 60.6±12.4 55.9±15.0 0.388aBMI (Kg/m2) 27.2±2.1 27.2±2.2 27.2±2.0 0.965aArterial hypertension (%) 11 (36.7) 6 (54.6) 5 (26.3) 0.238bDM (%) 8 (26.7) 1 (9.1) 7 (36.8) 0.199bComplications

Ascites (%) 12 (40.0) 5 (45.5) 7 (36.8) 0.712bHCC (%) 0 (0) 0 (0) 0 (0)UGB (%) 6 (20.0) 3 (27.3) 3 (15.8) 0.641bHRS (%) 0 (0) 0 (0) 0 (0)HE (%) 0 (0) 0 (0) 0 (0)

Child-Pugh grading 0.308cA (%) 16 (53.3) 5 (45.5) 11 (57.9)B (%) 12 (40.0) 4 (36.4) 8 (42.1)C (%) 2 (6.7) 2 (18.2) 0 (0)

BMI: body mass index; DM: diabetes mellitus; HCC: hepatocellular carcinoma; UGB: upper gastrointestinal bleeding; HRS: hepatorenal syndrome; HE: hepaticencephalopathy.p-value determined by a: Student’s t test; b: Fisher’s exact test; c: the Mann-Whitney U test.

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why we decided to integrate this risk factor as a necessarycriterion for diagnosing NASH-related LC in our study.Although the increases in NASH-related LC and obesity

may lead to a higher incidence of HCC, we unexpectedlyfound that the HCC incidence was significantly lower in thetime period from 2008-2013 than in the period from 2003-2005. Another recent study, which used data from the CancerRegistration Database records in Shanghai, China, also noteda decreasing HCC incidence (25). This study additionallyfound that the age-standardized incidence rates for each yearand the age-standardized mortality rates decreased from2006-2008 and that the incidence and mortality ratesincreased with age. The incidence and mortality rates ofprimary liver cancer were relatively higher in the suburbsthan in urban areas and were also higher in males than infemales. Additionally, a study from Taiwan found that malegender was a risk factor for HCC, which is in accordancewith our results based on univariate analysis. However, DMand obesity were not associated with HCC in areas whereeither HBV or HCV was endemic (26). In our study, obesityand DMwere not associated with HCC in the whole cirrhoticpopulation, in accordance with the Taiwanese study. How-ever, when considering only the HBV-related cirrhoticsubgroup, we observed a statistically insignificant trend ofincreased HCC in patients who were obese or diabetic. Theprevalence of NAFLD-related HCC is rising worldwide:4-22% of HCC cases in Western countries are now attributedto NAFLD (13). Additionally, 10-75% of cases of NAFLD-related HCC occur in patients without cirrhosis (27,28).A higher BMI in childhood increases the risk of primary livercancer in adults (29). In our study, HCC was not found inpatients with NASH-related LC, whereas HCC was presentin 31.5% of NASH-related LC cases in the Japanese

nationwide study (4). A study from England found thatNAFLD accounted for 41 (34.8%) cases of HCC in 2010 (30).Another study on HCC from Germany also found anincreasing proportion of NASH in HCC patients (2). Withthe promotion of an HBV vaccination program and theimprovement of economic development, this type of timetrend may also occur in China. Therefore, we should expandour attention from patients with cirrhosis caused by chronicviral hepatitis to those with cirrhosis induced by NASH inthe future. Additionally, patients with NASH-related LCmust be followed in the coming decades, paying specialattention to the effects in obese children, as the vaccinationprogram has protected them from HBV.For NASH-related LC, we found that female patients were

older than males and that the rates of complications andChild-Pugh grade C tended to be higher in women, which isin accordance with the Japanese nationwide study (4).However, the difference in our study was not significant,possibly due to the small sample size. The epidemiologicalstudy in Japan observed a higher NAFLD prevalence in menthan in women. However, in contrast to men, NAFLDincreased with age in women, showing a higher prevalenceamong women aged 40-49 years and after menopause (31).Aging is considered as a risk factor for NAFLD in pre-menopausal women (32). Our study showed an average ageof 60 years for women who developed NASH-related LC,indicating that they were menopausal women. Becauseestradiol has been reported to attenuate hepatic stellate cellactivation and therefore decrease the fibrotic process (33),estradiol diminution after menopause might contribute tothe development of NASH-related cirrhosis.Our study has several limitations. First, because the study

was retrospective, it was not possible to collect certain

Table 4 - Selected risk factors for HCC.

Variable Control HCC Univariate analysis Multivariate analysis

N=1,495 (%) N=87 (%) OR (95% CI) p-value OR (95% CI) p-value

Age

o65 1,227 (94.6) 70 (5.4) 0.93 (0.52-1.64) 0.796X65 268 (94.0) 17 (5.9)

SexFemale 468 (96.3) 18 (3.7) 1.81 (1.04-3.14) 0.036Male 1,027 (93.7) 69 (6.3)

ObesityObese 216 (94.3) 13 (5.7) 1.06 (0.58-1.96) 0.862Non-obese 1,279 (94.5) 74 (5.5)

HypertensionHypertensive 123 (95.3) 6 (4.6) 0.90 (0.37-2.21) 0.825Non-hypertensive 1,362 (94.4) 81 (5.6)

DMDiabetic 151 (95.0) 8 (5.0) 0.93 (0.43-1.99) 0.849Non-diabetic 1,344 (94.5) 79 (5.6)

HBs AgHBs Ag positive 1,008 (93.1) 75 (6.9) 2.64 (1.01-6.92) 0.049 3.35 (1.76-6.36) 0.000HBs Ag negative 487 (98.6) 7 (1.4)

Hepatitis B*HBV+obesity 121 (91.7) 11 (8.4) 1.31 (0.66-2.58) 0.436HBV+non-obesity 754 (93.6) 52 (6.5)HBV+DM 65 (92.8) 5 (7.1) 1.27 (0.53-3.07) 0.589HBV+non-DM 810 (93.3) 58 (6.7)

Alcohol#

HBV+alcohol abuse 123 (91.1) 12 (8.9) 0.74 (0.39-1.42) 0.364HBV+no alcohol abuse 885 (93.4) 63 (6.7)

*Cirrhotic patients with chronic liver disease caused only by HBV infection (938 patients).#Alcohol abuse in cirrhotic patients with HBs Ag positivity (1,083 patients).

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important metabolic parameters such as blood glucose for allcirrhotic patients, which may have led to underestimation ofthe rate of DM in LC. Second, histological data were rarelyobtained for our patients, which confined the diagnosis ofNASH-related cirrhosis to clinical, biochemical, and imagingdata. However, it should be recalled that when cirrhosis ishistologically present, the etiological features may disappear,so a liver biopsy may not be able to ascertain NASH-relatedcirrhosis. Third, the relatively small number of NASH-related LC cases might explain the absence of significantdifferences when considering important risk factors reportedin other studies. Finally, the history of NAFLD and theduration of obesity or DM could not be obtained from thedatabase.In conclusion, HBV remains the main etiology of LC in

China. However, NASH-related LC has clearly increased infrequency in recent years. Further studies are warranted toevaluate the exact importance of obesity and diabetes inHCC development in the overall cirrhotic population.

’ ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation ofChina (Grant No. 81170382 and 81200297).


Xiong J designed the questionnaire for data collection from the database,was responsible for the data entry and statistical analysis, and wrote the firstversion of the manuscript. Wang J collected data from the database andparticipated in data entry. Huang J collected data from the database andparticipated in data entry. Sun W checked the data and collected referencesfor this manuscript. Wang J revised the manuscript. Chen D participated inthe overall process of writing and revising the manuscript and served as thecorresponding author.

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Glutamine treatment attenuates hyperglycemia-inducedmitochondrial stress and apoptosis in umbilical veinendothelial cellsSher Zaman Safi,I,* Kalaivani Batumalaie,I Marzida Mansor,II Karuthan Chinna,III Syam Mohan,IV

Hamed Karimian,IV Rajes Qvist,I Muhammad Aqeel Ashraf,V Garcie Ong Siok YanII

IUniversity of Malaya, Faculty of Medicine, Department of Medicine, Kuala Lumpur, Malaysia. IIUniversity of Malaya, Faculty of Medicine, Department of

Anesthesiology, Kuala Lumpur, Malaysia. IIIUniversity of Malaya, Faculty of Medicine, Department of Social and Preventive Medicine, Kuala Lumpur,

Malaysia. IVUniversity of Malaya, Faculty of Medicine, Department of Pharmacy, Kuala Lumpur, Malaysia. VUniversity of Malaya, Faculty of Science,

Department of Geology, Kuala Lumpur, Malaysia.

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis,mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilicalvein endothelial cells.

MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamineand insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and theproduction of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicityassays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay.

RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability andcytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level wassignificantly (po0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealedsignificantly reduced apoptosis (po0.005) in the glutamine-treated cells. Moreover, glutamine alone or incombination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascularendothelial growth factor were up-regulated while tumor necrosis factor-a was down-regulated aftertreatment with glutamine.

CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrialstress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression ofinflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner.

KEYWORDS: Hyperglycemia; Sepsis; Apoptosis; Cytokine; Glutamine.

Safi SZ, Batumalaie K, Mansor M, Chinna K, Mohan S, Karimian H, et al. Glutamine treatment attenuates hyperglycemia-induced mitochondrialstress and apoptosis in umbilical vein endothelial cells. Clinics. 2015;70(8):569-576

Received for publication on April 29, 2015; First review completed on June 9, 2015; Accepted for publication on June 9, 2015



’ INTRODUCTION

Hyperglycemia is the abnormality underlying diabetesand several other complications. Chronic hyperglycemiacondition initiates a wide range of complications, includingcardiovascular disease, which is the most frequent cause ofdeath in the diabetic population (1,2). Hyperglycemia alsoinduces the production of reactive oxygen species (ROS) and,ultimately, DNA damage and apoptosis (3). Apoptosis has

been observed in the vascular cells, myocardium, and nervesof diabetic experimental animals and human subjects,although whether it contributes to or is a marker forcomplications in these tissues is unclear. Vascular diseasesare the major cause of morbidity and mortality in diabeticpatients. Several studies suggest that the endothelium is theinitial site where these vascular complications develop (4).During critical illness, hyperglycemia alone or hyperglyce-mia coupled with a relative insulin deficiency may directly orindirectly yield a predisposition to a spectrum of complica-tions, such as multi-organ failure and death (5-7). Morpho-logical correlates of these functional abnormalities were notinitially identified; however, several later studies showed anincrease in apoptosis in several organs affected by diabetesand sepsis, including the eye (8,9), heart, and vascularendothelium (10,11).DOI: 10.6061/clinics/2015(08)07


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mailto:[email protected]

mailto:[email protected]

Vascular endothelial cells are among the first cells in thebody to interact with bacterial endotoxins during sepsis.Sepsis is a very complex and heterogeneous clinicalcondition that is associated with hyperglycemia and insulinresistance (12). Vascular endothelial cells possess mechan-isms that recognize structural patterns of bacterial constitu-ents and initiate the expression of proinflammatory andanti-inflammatory pathways, which are tightly controlledto maintain homeostatic balance (13). However, in severesepsis, external stimuli, such as a severe infection, canactivate the immune cells and unleash a systemic inflamma-tory response that is expressed through various pathways(14). There is also increasing evidence to suggest that celldeath by apoptosis plays an important role in the pathogen-esis of severe sepsis/septic shock (15).Several studies have shown that endothelial cells can

undergo apoptosis in response to sepsis-related factors suchas Lipopolysaccharide (LPS) and Tumor necrosis factor alpha(TNF-a) (16-18). In addition, studies using in vitro models ofinfection have demonstrated that certain organisms arecapable of inducing endothelial apoptosis (19). Along withthe interaction of inflammation with apoptosis in sepsis,mitochondrial dysfunction seems to have a major impact insepsis patients because it has been closely linked to pro-grammed cell death. Alterations in mitochondrial functionhave been described in the muscle and liver mitochondriafrom septic rats and primates. Furthermore, mitochondrialdysfunction has been suggested as a potential mechanism toexplain tissue hypoxia despite normal oxygen availabilityduring sepsis (20,21). Pro-inflammatory cytokines, such asinterleukin 6 (IL-6), TNF-a, and other molecules, are releasedduring acute inflammation and result in endothelial activationand a significant increase in the expression of endothelialleukocyte adhesion molecule 1, vascular cell adhesion mole-cule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1),and vascular endothelial growth factor (VEGF). These proteinspromote leukocyte rolling, adherence, and migration, whichinitiate inflammation in the endothelium and other cells(22,23). We included IL-10 as an anti-inflammatory marker.Therefore, the aim of this study was to determine themechanism of endothelial cell apoptosis and the expressionof inflammatory cytokines under hyperglycemic conditionsand to examine the effects of glutamine and insulin.


Cell cultureEndothelial cells were obtained from VEC Technologies

(New York, USA). The cells were thawed at 37 ˚C andcultured in T25 flasks coated with 50 mg/ml of fibronectin.The cells were immersed in 5 ml of complete medium(MCDB-B-131), supplemented with 10% FBS, 1% penicillin-streptomycin, and epidermal growth factor (EGF, 10 ng/ml).The cells were incubated at 37 ˚C with 5% CO2. Trypsin/EDTA (1 ml for each flask) was used to detach the cells uponconfluency. All the experiments were performed at passages2-5.

Cell treatmentThe cells were seeded at 1x104 cells in each well and

incubated for 24 hours. Various concentrations of glucose,ranging from a normal value (5 mM) to a hyperglycemiclevel (20 mM), were added to the individual wells. Thehyperglycemic cells (glucose concentration 20 mM) were

divided into three groups. In the first group, 40 mM of gluta-mine was added. In the second group, 1.0 x 10-6 units/ml ofinsulin was added. In the third group, glutamine (40 mM)and insulin (1.0 x 10-6 units/ml) were added. The cells werethen incubated for the required length of time (24 hours). Forthe cytokine and TUNEL analyses, 0.7x106 cells were grownin T25 flasks using the same treatment groups. The cells wereharvested and frozen until required for analysis.

Western blottingThe endothelial cells were first lysed in cold lysis buffer

containing 20 mmol/l of TRIS HCl, 140 mmol/l of NaCl, 1mmol/l of EDTA and complete miniprotease inhibitor cock-tail, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1mmol/l NaF, and 1 mmol orthovanadate. The proteins (30 mg)were then loaded on 10% SDS polyacrylamide gels andtransferred to activated nitrocellulose membranes. The mem-branes were blocked with Tris-buffered saline (TBS) contain-ing 5% nonfat milk and incubated overnight with the primaryantibodies to IL-10 and TNF-a, obtained from Santa Cruz, at4 ˚C. Beta-actin was used as a loading control. After extensivewashes in TBS, the membranes were incubated for one hour atroom temperature with the appropriate horseradish perox-idase-conjugated secondary antibodies, and the proteins werevisualized using a chemiluminescence substrate according tothe manufacturer’s instructions (Amersham Life Sciences).

Multiple cytotoxicity assaysThe Cellomics Multiparameter Cytotoxicity 3–kit was used

as previously reported in detail by Cheah et al. (24). TheMultiparameter Cytotoxicity 3–kit enables parallel measure-ments of six independent parameters that monitor cellhealth, namely, changes in cell permeability, cell loss andnuclear size; changes in mitochondrial membrane potential;cytochrome c release; and morphological changes. Briefly, thecells were plated at 1x104 cells per well for 24 hours. Glucose(5 or 20 mM), glutamine (40 mM), and insulin (1.0 x 10-6

units/ml) were added in different combinations as describedin the cell treatment section, and the incubation wascontinued for 24 hours. The MMP dye and the cellpermeability dye were added to the live cells, and the cellswere incubated for 1 hour. The cells were fixed, permeabi-lized, and blocked with 1� blocking buffer before they wereincubated with the primary cytochrome c antibody andconjugated secondary antibody for 1 hour. The cells wererinsed three times with wash buffer II, and the plates wereanalyzed using the Array Scan HCS high content system(Cellomics, PA, USA).

Measurement of transmembrane mitochondrialpotential

The mitochondrial transmembrane potential results fromthe asymmetric distribution of protons and other ions on thetwo sides of the inner mitochondrial membrane, which givesrise to the chemical, pH, and electric gradients that areessential for mitochondrial function. The inner side of theinner mitochondrial membrane is negatively charged. Con-sequently, cationic lipophilic fluorochromes are distributedwithin the mitochondrial matrix as a function of the Nernstequation describing the transmembrane mitochondrialpotential (c). Using a cytofluorometer, these dyes wereemployed to measure variations in the transmembranepotential (c) on a per-mitochondrion or per-cell basis.

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Terminal deoxynucleotidyl transferase dUTP nickend-labeling (TUNEL) assayDNA damage was investigated using a 96–well colori-

metric apoptosis detection kit (R&D System) according to themanufacturer’s instructions. Umbilical vein endothelial cellswere cultured and transferred to a 96-well plate (1x105 cells/well). The cells were then fixed with 3.7% bufferedformaldehyde for 5 minutes, followed by washing withPBS. The washing was followed by permeabilization of thecells with 100% methanol for 20 minutes and another washwith PBS. Following the manufacturer’s protocol, the cellswere then subjected to the labeling procedure, and thereaction was stopped with 0.2 N HCl after 30 minutes. Thecells were treated with NucleaseTM to generate DNA breaksand to confirm the permeabilization and labeling reactions.An unlabeled control was included to indicate the level ofbackground labeling associated with non-specific binding ofthe Strep-HRP. The absorbance at 450 nm was measuredusing a microplate reader.

Cytokine measurementsThe cytokines TNF-a, IL-6, and IL-10 were measured in

triplicate using the Protein Bio-Plex Cytokine Assay (Bio-RadLaboratories). T25 flasks containing 0.7x106 cells werecultured, and the lysate was filter-sterilized (0.22-mm poresize). The protein concentrations were determined, and the

Bio-Plex Cytokine Assay (Bio-Rad Laboratories) was con-ducted according to the manufacturer’s protocol. Thecalculated concentrations for each cytokine were averaged,and the standard deviations were determined. Statisticalsignificance was determined using the t-test, where po0.05designated increased/decreased cytokine production in thepresence of glutamine or glutamine in combination withinsulin.

Statistical analysisEach experiment was performed at least two times.

Statistical analysis was performed using one-way analysisof variance (ANOVA).

’ RESULTS

Glutamine reduces the cytochrome C levels andapoptosis in hyperglycemic human umbilical veinendothelial cellsUmbilical vein endothelial cells were stained with Hoechst

in the presence of glucose (20 mM) alone, 20 mM glucose +40 mM glutamine, 20 mM glucose + 10-6 M insulin, or 20mM glucose + 40 mM glutamine + 10-6 M insulin, and thestaining intensity was determined. As shown in Figures 1and 2, glucose alone (20 mM) reduced the number of cells,possibly by apoptosis, as well as reduced the level of

Figure 1 - Representative images of endothelial cells treated with medium alone (control), glucose alone (20mM), glucose (20mM) +insulin (10-6 M), glucose (20mm) + glutamine (40mM) and glucose (20mM) + glutamine (40mM) + insulin (10-6 M). Cells were stainedwith Hoechst for nuclear, cell permeability dye, mitochondrial membrane potential dye and cytochrome c. The image from each rowwas obtained from the same field of the same treatment sample (magnification 20 xs).

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cytochrome c. There was a significant change (po0.005) inthe fluorescence intensity in the cells that were treated withglucose along with glutamine. This result indicates that thesecells were more viable. In addition, the cytochrome c levels inthe endothelial cells treated with glucose (20 mM) +glutamine (40 mM) were significantly (po0.005) lower thanthe levels in the cells treated with glucose (20 mM) alone. Thecytochrome c levels in the cells treated with glucose (20 mM)+ insulin (10-6 M) and glucose (20 mM)+ insulin (10-6 M)+glutamine (40 mM) also changed significantly (po0.05)compared with the levels in the cells treated with glucose(20 mM) alone (Figures 1 and 2B). Similarly, the TUNELassay revealed a significantly reduced level of apoptosiswhen the hyperglycemic cells were treated with insulin(po0.05), glutamine (po0.005), or glutamine + insulin(po0.005) (Figure 2A).

Glutamine restores the loss of mitochondrialmembrane potential (MMP) in Human UmbilicalEndothelial Cells (HUVEC)

According to previous studies, a reduction in themitochondrial membrane potential occurs when cellsundergo oxidative stress and hyperglycemia in thepresence of other co-factors. The cells showed a signifi-cant change in the MMP when treated with glutamineand a considerable, but not significant, change in theMMP when they were treated with glucose + glutamine+ insulin compared with 20 mM glucose only. Glucose(20 mM) + insulin (10-6 M) and glucose (20 mM) + insulin(10-6 M) + glutamine (40 mM) also yielded an increase in theMMP (DCm) compared with the control. These results showthat the MMP was reduced after the cells had been treated

Figure 2 - Shows (A) TUNEL assay which revealed a significantly reduced apoptosis when hyperglycemic cells were treated with insulin(po0.05) glutamine (po0.005) and glutamine + insulin (po0.005). (B) Cytochrome c intensity for the endothelial cells treated withglucose (20 mM) + glutamine (40 mM) significantly changes when compare to glucose (20 mM) alone. Cytochrome c intensity for thecells treated with glucose (20 mM) + insulin (10-6 M) and glucose (20 mM) + insulin (10-6 M) + glutamine (40 mM) changesinsignificantly as compare to glucose (20 mM) alone. (C) and (D) show mitochondrial membrane potential and cell permeabilityrespectively.

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with 20 mM glucose alone; however, glucose (20 mM) incombination with glutamine (40 mM) was shown to be moreprotective against the fall in the MMP and oxidative stress-induced cell death (Figure 2C).

Effect of glutamine on cell permeabilityWhen the MMP decline reaches a certain point, the

opening of the permeability transition pore (PTP) starts tocause extensive cell damage and, consequently, cell death.The hyperglycemia-induced permeability was reversedwhen we treated the HUVECs with glucose + glutamineor glucose + glutamine + insulin compared with glucose(20 mM) alone. However, there was a slight increase in thecell permeability when the cells were treated with glucose (20mM) + insulin (10-6 M) (Figure 2D).

Glutamine and inflammatory cytokinesWhen glucose (20 mM) and insulin (10-6 M) were added to

the culture, there was no difference in the expression of IL-6.However, glucose + glutamine significantly increased theexpression of IL-6. The combined treatment with glucose(20 mM), glutamine (40 mM), and insulin (10-6 M) alsosignificantly increased the expression of IL-6 (po0.005).Similarly, treatment with insulin alone did not alter theexpression of IL-10, but insulin in combination withglutamine increased the expression of IL-10 in the endothe-lial cells. A small increase in the expression of VEGF wasnoted while a reduced expression of TNF-a was observedwhen the cells were treated with the same concentration ofglutamine alone or in combination with insulin (Figure 3A,3B, 3C, and 3D). Detailed values of all cytokines are given inTable 1. IL-10 is the most important anti-inflammatory

Figure 3 - (A) shows that insulin did not alter the expression of IL–10 when treated alone but insulin in combination with glutamineincreased IL–10 in endothelial cells (po0.05). (B) Shows a significantly reduced TNF–a when cells were treated with the sameconcentration of glutamine in combination with insulin. (C) IL–6 expression which is significantly increased when treated withglutamine in combination with insulin (po0.005). (D) A mild increase in the expression of VEGF was noted when treated withglutamine or glutamine in combination with insulin.

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cytokine found within the human response. It is a potentinhibitor of Th1 cytokines and a potent activator ofmonocyte/macrophage proinflammatory cytokine synthesis.In addition, IL-10 attenuates the surface expression of TNFreceptors and promotes the shedding of the TNF receptorsinto the systemic circulation. Furthermore, there is aninteresting relationship between IL-10 and the soluble TNF-a receptor. Specifically, when IL-10 increases, it causes anincrease in the levels of the soluble TNF-a receptor, whichresults in decreased TNF-a levels. To confirm this effect, we

carried out Western blotting for IL-10 and TNF-a (Figure 4).The levels of both IL-10 and TNF-a changed in the samemanner, as shown in Figure 3.

’ DISCUSSION

Our results show that glutamine, either alone or incombination with insulin, disrupts mitochondrial stress andimproves cell viability. Anti-inflammatory cytokines were highlyexpressed in the glutamine-treated cells. With respect tocytochrome C, there was a significant change in the fluorescenceintensity in the cytosol in the cells treated with glutamine alone,indicating an increase in the viability of the cells (Figure 2B) onthe basis that increased cytochrome c levels in the cells triggerprogrammed cell death through apoptosis (25,26). Similarly,there was an increase in the mitochondrial potential (Figure 2C).

Our results demonstrate that the cytochrome c levels in cellsare increased under hyperglycemic conditions. This protein isknown for its function in the mitochondria as a key participantin the life-supporting function of ATP. Our data also support thehypothesis that hyperglycemia, through the production ofoxidative stress, could be an apoptotic stimulus that triggersthe release of cytochrome c into the cytosol, thereby activatingthe mitochondrial pathway that leads to the permeabilization ofthe outer mitochondrial membrane and increasing the level ofcytochrome c (27,28). In the cytosol, cytochrome c engagesapoptotic protease activating factor-1 (APAF1) and forms theapoptosome. The cell then dies via the apoptotic pathway ornecrosis due to the collapse of electron transport, generation ofoxygen free radicals, and production of ATP (29,30). As shownin Figure 2B, in our study, the level of cytochrome c in cells

Figure 4 - (A) shows the effect of insulin and glutamine on the expression of IL–10 and TNF–a in endothelial cell. (B) shows significantlyreduced IL–10 and (C) shows TNF–a in graphical form.

Table 1 - Cytokine analysis comparing 20 mM glucose, 20 mMglucose + Ins, 20 mM glucose + Gln, and 20 mM glucose + Ins+ Gln.

Variable Group n Mean p-value

Hu IL-6 20 mM 3 55.520 mM+Ins 3 56.2 NS20 mM+Gln 3 73.7 0.03520 mM+Ins+Gln 3 91.0 0.004

Hu IL-10 20 mM 3 26.320 mM+Ins 3 27.5 NS20 mM+Gln 3 32.2 NS20 mM+Ins+Gln 3 34.8 0.011

Hu TNF-a 20 mM 3 13.320 mM+Ins 3 12.8 NS20 mM+Gln 3 10.7 NS20 mM+Ins+Gln 3 9.7 0.006

Hu VEGF (45) 20 mM 3 22.020 mM+Ins 3 20.7 NS20 mM+Gln 3 22.5 NS20 mM+Ins+Gln 3 24.7 NS

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cultured in 20 mM glucose (hyperglycemic conditions) wassignificantly reduced by the addition of glutamine (40 mM), andthe viability of the cells was thus increased compared with thatof cells incubated with insulin alone or insulin plus glutamine.Cells induced to undergo apoptosis show an early

reduction in the incorporation of c-sensitive dyes, whichindicates a decrease in the transmembrane potential. Thistransmembrane potential disruption can be detected in manydifferent cell types, irrespective of the apoptosis-inducingstimulus. The transmembrane potential disruption occursbefore the cells exhibit nuclear DNA fragmentation, indicat-ing that the membrane potential change constitutes an earlycommon event of the apoptotic cascade. Purified cells with alow transmembrane potential rapidly proceed to DNAfragmentation. In our study, there was a decrease in themitochondrial potential under the hyperglycemic condition,suggesting that hyperglycemia could act as an apoptosis-inducing stimulus. The decrease in the mitochondrialpotential was reduced in the presence of glutamine orglutamine plus insulin (Figure 2C), although this reductiondid not reach significance in the case of glutamine andinsulin combined. An intact transmembrane potential (c) isindispensable for normal mitochondrial function becausecells undergoing apoptosis cease mitochondrial biogenesis atboth the translational and transcriptional level (31). More-over, during apoptosis, mitochondrial inner membraneproteins, including cytochrome c, leak out into the cytosol.Mitochondria play a central role in the apoptotic process,

in which the dissipation of MMP, increased mitochondrialoxidant production, and release of apoptogenic proteins(e.g., apoptosis-inducing factor and cytochrome c) causedby opening of the permeability transition pore are observed.In our study, the cell permeability was increased underhyperglycemic conditions, but the permeability was reducedin the cells treated with glutamine (Figure 2D).Recent evidence (32) has implicated a general dysregula-

tion of the endothelium, with apoptosis and necrosis as thefinal pathway of endothelial dysfunction and with mito-chondrial dysfunction caused by the central disruption ofcellular oxidative function. We therefore hypothesized thatmitochondrial dysfunction may also be present in endothe-lial cells during hyperglycemia and may reflect the degree ofsystemic injury in patients with severe sepsis and hypergly-cemia. Stress-induced hyperglycemia and insulin resistanceare exceedingly common in critically ill patients, particularlyin those with sepsis. Multiple pathogenic mechanisms areresponsible for this metabolic syndrome, with the increasedrelease of proinflammatory mediators and counter-regula-tory hormones playing a pivotal role (33). Recent datasuggest that hyperglycemia may potentiate the proinflam-matory response, while insulin has the opposite effect.To investigate the possibility that hyperglycemia plays a

key role in the development of the inflammatory response insepsis, we assessed the patterns of IL-6, TNF-a, VEGF, andIL-10 that are associated with severe sepsis. In an exploratoryanalysis, one study (34) demonstrated that by using multiplecytokine assays, distinct cytokine profiles were found to beassociated with the severity of sepsis, the development oforgan failure, and death. The inflammatory cytokines IL-1b,IL-6, IL-8, IL-10, and TNF-a have been shown to beassociated with the various stages of severe sepsis. Todetermine whether the same group of cytokines wasexpressed under hyperglycemic conditions, we assessed theexpression of IL-6, TNF-a, VEGF, and IL-10. In our study, we

determined the effect of insulin and glutamine on theexpression of IL-6, a cytokine with anti–inflammatory andproinflammatory functions. When insulin was added to thecultures, there was no difference in the expression ofIL-6 (Figure 3), but glutamine had an additive effect withinsulin, and the combination of glutamine and insulinsignificantly increased the expression of IL-6. Like manyother cytokines, IL-6 has both pro–inflammatory and anti-inflammatory properties. Recent evidence generated usingIL-6 knockout mice has demonstrated that IL-6, like othermembers of the gp130 receptor ligand family, acts predomi-nantly as an anti–inflammatory cytokine. IL-6 down-reg-ulates the synthesis of IL-1 and TNF-a and attenuates thesynthesis of pro–inflammatory cytokines. Simultaneously,IL-6 inhibits the production of pro–inflammatory cytokines,including GM–CSF, IFNg, and MIP2. Interestingly, IL-6 maydown-regulate TNF-a and IL-b production and may beimportant in limiting the inflammatory response. Our resultsdemonstrate the same limiting response of IL-6 on TNF-a;however, VEGF was unexpectedly up-regulated by theglutamine treatment. The net results of these immunologiceffects place IL-6 among the anti-inflammatory group (35).The expression of IL-10 (Figure 3) was increased by

treatment with glutamine and insulin, with the addition ofinsulin having an additive effect. IL-10 is the most importantanti-inflammatory cytokine in humans. It is a potentinhibitor of the Th1 cytokines and a potent deactivator ofmonocyte/macrophage pro-inflammatory cytokine synth-esis. In addition, IL-10 attenuates the surface expression ofTNF receptors and promotes the shedding of the TNFreceptors into the systemic circulation (35-37).The above cytokines have both anti-inflammatory and pro–

inflammatory functions (38). Therefore, even during aninflammatory disease such as sepsis, it is important to maintainboth the inflammatory and anti-inflammatory cytokines, andthis balance seems to be achievable by the effect of glutamine.According to previous reports, glutamine supplementation hasbeen shown to maintain the T–lymphocyte population in thespleen and to significantly enhance the mRNA expressionlevels of Th1 and Th2 cytokines and TNF-a in the spleens ofrats with septic peritonitis (39,40).Pharmacological supplementation with glutamine helps to

maintain the intestinal barrier, modulate cytokine production,and prevent organ injury during sepsis. However, the exactprotective mechanism remains to be explored. It has alreadybeen demonstrated that glutamine significantly attenuates theplasma levels of cytokines produced by macrophages andendothelial cell necrosis after cecal ligation and puncture in rats(41,42). Recently, it was reported that glutamine treatmentdirectly augmented macrophage TNF-a production in vitro butdecreased TNF-a release in vivo, even though the expression ofHSP72 was increased in both cases (43). Another reportsuggests that dietary glutamine administration results in higherinflammatory cytokine production and greater neutrophilrecruitment during the early stage of acute lung injury (44).In conclusion, our data suggest that glutamine alone or in

combination with insulin can modulate the production ofinflammatory and anti-inflammatory cytokines and maintainthe cytokine balance under hyperglycemic conditions,producing a cytoprotective effect. At the same time, ourdata indicate that glutamine maintains the integrity of themitochondria, the dysfunction of which in hyperglycemicendothelial cells may reflect the degree of systemic injury insevere sepsis.

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’ ACKNOWLEDGEMENTS

This work was supported in part by grants (No. RG074/09AFR, and RG528-13HTM (UMRG)) from the University of Malaya. We thank ArokiasamyVinsent Rayappan (Department of Medicine, Faculty of Medicine, UM) forhelping in the cell culture work. We declare there is no conflict of interest.


Safi SZ performed the basic work and wrote the manuscript. Batumalaie Khelped with the lab work. Karimian H helped with the reagents. Mansor M,Mohan S, Qvist R, and Yan GOS designed the study and reviewedthe manuscript several times. Chinna K and Ahraf MA helped with thestatistical analysis.

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http://dx.doi.org/10.1097/00075197-200303000-00011


Effect of hypertonic saline treatment on theinflammatory response after hydrochloric acid-inducedlung injury in pigsCarla Augusto Holms,I Denise Aya Otsuki,I,* Marcia Kahvegian,I Cristina Oliveira Massoco,II

Denise Tabacchi Fantoni,III Paulo Sampaio Gutierrez,IV Jose Otavio Costa Auler JuniorI

I Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Anesthesiology (LIM-08), Sao Paulo/SP, Brazil. II Faculdade de Medicina Veterinaria e

Zootecnia da Universidade de Sao Paulo, Department of Pathology. IIIDepartment of Surgery, Sao Paulo/SP, Brazil. IV Instituto do Coracao do Hospital das

Clınicas da Faculdade de Medicina da Universidade de Sao Paulo, Laboratory of Pathology, Sao Paulo/SP, Brazil.

OBJECTIVES: Hypertonic saline has been proposed to modulate the inflammatory cascade in certainexperimental conditions, including pulmonary inflammation caused by inhaled gastric contents. The presentstudy aimed to assess the potential anti-inflammatory effects of administering a single intravenous dose of7.5% hypertonic saline in an experimental model of acute lung injury induced by hydrochloric acid.

METHODS: Thirty-two pigs were anesthetized and randomly allocated into the following four groups: Sham,which received anesthesia and were observed; HS, which received intravenous 7.5% hypertonic saline solution(4 ml/kg); acute lung injury, which were subjected to acute lung injury with intratracheal hydrochloric acid; andacute lung injury + hypertonic saline, which were subjected to acute lung injury with hydrochloric acid andtreated with hypertonic saline. Hemodynamic and ventilatory parameters were recorded over four hours.Subsequently, bronchoalveolar lavage samples were collected at the end of the observation period to measurecytokine levels using an oxidative burst analysis, and lung tissue was collected for a histological analysis.

RESULTS: Hydrochloric acid instillation caused marked changes in respiratory mechanics as well as blood gas andlung parenchyma parameters. Despite the absence of a significant difference between the acute lung injury andacute lung injury + hypertonic saline groups, the acute lung injury animals presented higher neutrophil andtumor necrosis factor alpha (TNF-a), interleukin (IL)-6 and IL-8 levels in the bronchoalveolar lavage analysis. Thehistopathological analysis revealed pulmonary edema, congestion and alveolar collapse in both groups;however, the differences between groups were not significant. Despite the lower cytokine and neutrophil levelsobserved in the acute lung injury + hypertonic saline group, significant differences were not observed amongthe treated and non-treated groups.

CONCLUSIONS: Hypertonic saline infusion after intratracheal hydrochloric acid instillation does not have aneffect on inflammatory biomarkers or respiratory gas exchange.

KEYWORDS: Acute lung injury; Hypertonic saline; Pigs; Hydrochloric acid; Inflammation.

Holms CA, Otsuki DA, Kahvegian M, Massoco CO, Fantoni DT, Gutierrez OS, et al. Effect of hypertonic saline treatment on the inflammatoryresponse after hydrochloric acid-induced lung injury in pigs. Clinics. 2015;70(8):577-583

Received for publication on May 5, 2015; First review completed on May 19, 2015; Accepted for publication on May 19, 2015



’ INTRODUCTION

The aspiration of acid gastric contents is the second mostcommon cause of direct acute lung injury (ALI) (1), and itaccounts for 10% of all acute respiratory distress syndrome(ARDS) cases (2). Pulmonary inhalation of gastric contentsmay provoke ALI with different degrees of respiratory

compromise and is frequently observed among patientswho have depressed airway protective reflexes, such astraumatized patients (3,4). In addition to the potential riskof developing ARDS (5), the inhalation of gastric contentsis a common cause of donor lung rejection (6).Hyperosmolar solutions, such as hypertonic saline, have been

tested in an attempt to reduce inflammation in ALI (7-9).However, previous results are contradictory and indicate thatthe effects of hypertonic saline depend on the type and agent oflung injury and timing of administration (10,11).The ALI model used in the current study involves the

intratracheal instillation of hydrochloric acid (HCl) andattempts to mimic the clinical scenario of gastric contentaspiration, wherein a pulmonary lesion is caused by the actionDOI: 10.6061/clinics/2015(08)08


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BASIC RESEARCH

of HCl. The acid causes chemical lesions in the lungs, resultingin diffuse injury to the alveoli, release of inflammatorymediators, recruitment of polymorphonuclear cells and promo-tion of severe edema, and these complications cause asignificant reduction in pulmonary compliance and compro-mise gas exchange (12-14). We hypothesized that hypertonicsaline would modulate HCl-induced lung injury. The purposeof this investigation was to test whether a 7.5% hypertonicsaline dose could attenuate the inflammatory response evokedby HCl instillation.

’ METHODS

This study was approved by the Ethics and AnimalInvestigation Committee of the affiliated institution (CAPPesq0363/08) and was performed in accordance with the Guide forthe Care and Use of Laboratory Animals (15). Thirty-twoyoung female Landrace pigs weighing between 27 and 33 kg(29±2.9) were included in this study. Animals were submittedto 12-hour fasting with free access to water, pre-medicatedintramuscularly with ketamine (5 mg/kg) and midazolam(0.5 mg/kg), and catheterized using a marginal ear vein fordrug and fluid administration. After sedation, the animals wereanesthetized with an intravenous dose of propofol (3-5 mg/kg), orally intubated (6.5 mm internal diameter cuffed endo-tracheal tube) and placed in the supine position. The lungswere mechanically ventilated (Primus, Dräger, Germany) usingpressure-controlled ventilation (FiO2 50%, tidal volume of8 ml/kg, inspiratory:expiratory (I:E) of 1:2 and positive end-expiratory pressure (PEEP) of 5 cmH2O), with the respira-tory frequency adjusted to maintain end-tidal CO2 between35 and 40 mmHg. To achieve patient-ventilator synchrony,pancuronium bromide (0.3 mg/kg/h) was administeredthrough continuous venous infusion. Anesthesia was main-tained with 1.5% isoflurane administered through a cali-brated vaporizer (Vapor 2000, Draeger, Lubeck, Germany).The body temperature was maintained between 37 ˚C and

38 ˚C using a circulating water mattress.

MonitoringAn arterial catheter was inserted into the right femoral

artery, and a 7.5 French pulmonary artery catheter thatmeasured continuous cardiac output (Edwards LifesciencesCorp., Irvine, CA) was inserted into the right jugular vein.The heart rate (HR), mean arterial pressure (MAP), mean

pulmonary artery pressure (MPAP), pulmonary arterywedge pressure (PAWP) and central venous pressure (CVP)were obtained directly using a multiparametric monitor(IntelliVue MP40, Phillips, Böblinger, Germany). Using

conventional formulae, following indexes were derived:cardiac index (CI), pulmonary vascular resistance index(PVRI) and systemic vascular resistance index (SVRI).

The inspiratory peak pressure, plateau inspiratory pres-sure, static pulmonary compliance, respiratory frequencyand tidal volume were obtained directly from the ventilatormonitor. Arterial blood samples were collected at each timepoint and were immediately analyzed (ABL 555; Radiometer,Copenhagen, Denmark), and the PaO2/FiO2 ratio wascalculated.

Study designAfter preparation, all of the animals were submitted to

three series of recruitment maneuvers consisting of 30 s ofsustained inflation with 20 cmH2O pressure followed by 30 sof regular ventilation. The animals were allowed to stabilizefor 30 min and then randomly assigned to four groups: Sham(n=8), hypertonic saline (HS; n=8), acute lung injury (ALI;n=8) and acute lung injury + hypertonic saline (ALI+HS;n=8). ALI was induced in the ALI and ALI+HS groups viaintratracheal instillation of HCl through the distal port of abronchoscope. The ALI+HS animals were treated with 7.5%hypertonic saline (4 mg/kg) 15 min after HCl instillation.The Sham and HS groups served as controls. The animalsfrom the HS group were administered 7.5% hypertonic saline(4 ml/kg) 15 min after the baseline measurement.

The experimental protocol is outlined in Figure 1.

Collecting pointsFollowing a 30-min stabilization period, baseline (BL)

measurements were performed. ALI was induced immedi-ately after BL measurements in the ALI and ALI+HSgroups. A new series of measurements was collected 30 minafter the administration of HCl (T30) and hourly thereafter(T90, T150, T210, T270).

Blood samples were collected at the above time points, andbronchoalveolar lavage (BAL) sampling was performed with3 x 20 ml phosphate buffered saline (PBS) at T270 throughbronchoscopy of the upper right lobe of the lung.

ALI induced by HClA 0.1 N HCl pH 1.0 solution was prepared by the

hospital’s pharmacy department and instilled at a dose of4 ml/kg body weight at the level of the carina over a 4 minperiod through a bronchoscope (Pentax FB-15H, Montvale,NJ). ALI was established when the PaO2/FiO2 ratio fellbelow 300 mmHg, which was achieved approximately 10min after hydrochloric acid inhalation.

Figure 1 - Study design.

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CLINICS 2015;70(8):577-583

Neutrophil count and oxidative burst analysisThe BAL samples were pooled, and a 0.2 ml aliquot was

transferred to an Eppendorf tube, stained with 0.2% trypanblue solution and counted using a Neubauer chamber.As previously described (16,17), oxidative burst analysis

was performed using a flow cytometer (Becton DickinsonImmunocytometry System, San Jose, CA, USA) connected toa computer (Apple, Fremont, CA, USA). BAL cells (2 x 105

cell) were incubated in a 37 ˚C shaking water bath for 30 minwith 200 ml of dichloro-dihydro-fluorescein diacetate(DCFH-DA; 0.3 mM, Molecular Probes, Invitrogen, Carlsbad,CA, USA), 200 ml of DCFH-DA and 100 ml of phorbol-12-myristate-13-acetate (PMA; 1 ng/1 ml, Calbiochem, Gibbs-town, NJ, USA). In total, 10,000 cellular events wereanalyzed by Cell Quest Software, and the results wereexpressed as the geometric mean fluorescence intensity(GMFI).

Cytokine measurementsBAL samples (10 ml) were centrifuged (2,000 rpm, 10 min,

4 ˚C), and the supernatant was stored at -80 ˚C for subsequentanalysis. BAL cytokine levels (interleukin (IL)-1, IL-8, IL-10, andtumor necrosis factor alpha (TNF-a)) were assessed usingcommercially available immunoenzymatic assay (ELISA) kits

containing pig-specific monoclonal antibodies, according to themanufacturers’ instructions (R&D Systems, Minneapolis, MN,USA). The obtained concentrations were transformed intopg/ml values using a nonlinear regression curve.

Lung histologyAt the end of the study, the trachea was clamped at the end of

the inspiratory cycle, and the animal’s thorax was opened. Foursamples were collected from the middle area of the left apical(West’s zone 2) and diaphragmatic lung lobes as well as fromthe middle of the right apical and diaphragmatic lung lobes. Thesamples were fixed in a 10% formaldehyde solution forsubsequent histologic analysis. The tissue samples wereembedded in paraffin, and 5 mm histological sections werestained with hematoxylin and eosin. Two pathologists whowere blinded to the study groups performed the histologicalanalyses simultaneously. Examinations included testing for thepresence of edema, intra-alveolar and interstitial hemorrhagesand polymorphonuclear and mononuclear cell infiltration.Each assessed histological characteristic was attributed ascore from 0 to 3 according to the level observed in the tissue(absent (0), mild (1), moderate (2) or severe (3)). The final scorefor the animal was determined according to the sum of thescores from each lobe (maximum score 12).

Table 1 - Hemodynamics and respiratory variables in the control (SHAM and hypertonic saline) and acid lesion (acute lung injury andacute lung injury + hypertonic saline) groups.

Variable Group BL T30 T90 T150 T210 T270

CI (L/min/m2) SHAM 3.6±0.6 3.7±0.5 4.0±0.5 4.3±0.5 4.3±0.7 4.3±0.7HS 3.7±0.3 4.1±0.4 4.9±0.4*w 4.9±0.6* 4.6±0.7* 4.5±0.7*ALI 3.9±0.6 4.2±0.9 4.4±0.9 4.4±0.8 4.2±0.7 4.2±0.5ALI+HS 3.5±0.3 4.3±0.4* 4.8±0.5* 4.8±0.4* 4.8±0.4* 4.6±0.5*

HR (bpm) SHAM 94±8 97±11 101±19 104±15 107±21 108±28HS 104±15 105±11 115±15 129±18 122±18 117±10ALI 106±13 124±15 112±19 122±31 119±30 119±21ALI+HS 92±12 114±13 120±21 121±12 124±17 121±19

MAP (mmHg) SHAM 54±7 59±6* 69±10* 66±12* 61±9* 61±8*HS 67±3 72±4* 84±10* 81±12* 76±9* 77±7*ALI 70±8 80±16* 80±8* 84±9* 82±8* 81±5*ALI+HS 66±3 83±17* 83±11* 85±11* 85±12* 82±8*

MPAP (mmHg) SHAM 14±2 16±2 15±3 15±3 16±3 15±2HS 16±2 16±1 19±1* 16±6 18±2** 19±2*ALI 16±2 25±3*w# 19±3*w# 22±3*w# 21±3*w# 22±4*w#ALI+HS 14±3 26±5*w# 21±3*w# 24±3*w# 24±4*w# 23±3*w#

CVP (mmHg) SHAM 9±3 11±2 10±3 10±3* 10±2* 10±2HS 10±2 11±1 11±1 10±1* 10±2* 10±2ALI 10±1 9±1 10±1 10±1* 10±1* 10±1ALI+HS 9±1 11±1 9±1 10±1* 10±1* 10±1

PVRI (dyne.sec.cm�5.m2) SHAM 131±20 129±18 121±16 131±30 132±28 131±30HS 128±15 119±30 130±40 130±27 132±31 149±28*ALI 152±31 331±114*w# 225±51*w# 266±77*w# 230±58*w# 290±51*w#ALI+HS 137±35 364±127*w# 207±57*w# 266±53*w# 266±66*w# 263±58*w#

SVRI (dyne.sec.cm� 5.m2) SHAM 1317±188 1249±138 1348±158 1302±236 1362±202 1274±111HS 1185±124 1178±140 1157±202 1115±200 1116±228 1172±272ALI 1228±152 1343±335 1317±354 1372±320 1419±352 1375±187ALI+HS 1266±175 1349±291 1222±254 1247±280 1251±274 1241±269

PPlat (cmH2O) SHAM 13±1 14±1* 14±1.7* 14±1.6* 14±1.6* 14±1.6*HS 14±1 14±1,8 14±1.8* 15±1.6* 15±1.9* 15±2*ALI 14±1 23±1.3*w# 21±1.5*w# 21±1.7*w# 21±1.8*w# 21±1.7*w#ALI+HS 14±1 23±1.6*w# 21±1.7*w# 21±1.6*w# 21±1.2*w# 21±1.2*w#

Compl (cmH2O) SHAM 31±4.1 30±4.5 29±4 29±4.3* 28±4* 28±3.6*HS 29±4.5 28±4.6 26±3.8* 26±3.8* 25±4.4* 24±4.3*ALI 28±4.2 14±1.4*w# 16±2*w# 16±1.9*w# 16±1.5*w# 16±2.5*w#ALI+HS 28±4.2 13±2*w# 15±1.6*w# 15±1.7*w# 15±1.7*w# 15±2*w#

Data are expressed as the mean±standard deviation. CI: cardiac index; HR: heart rate; MAP: mean arterial pressure; MPAP: mean pulmonary arterialpressure; CVP: central venous pressure; PVRI: pulmonary vascular resistance index; SVRI: systemic vascular resistance index; PPlat: plateau pressure; Compl:pulmonary compliance; *po0.05 compared with the baseline; wpo0.01 compared with the Sham group; # po0.05 compared with the HS group.

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CLINICS 2015;70(8):577-583 Hypertonic saline in lung injuryHolms CA et al.

Statistical analysisThe results were expressed as the mean±standard

deviation (SD). Statistical analyses were performed using arepeated measures analysis of variance (ANOVA) followedby Tukey’s test to analyze the group effect on the investigatedparameters. A one-way ANOVA was used to analyze thehistopathological results. Significance was established at thelevel of 5% (po0.05).

’ RESULTS

The body weights of the animals did not differ betweengroups. In addition, the administered amount of infused7.5% hypertonic saline (HS: 171±16 ml; ALI+HS: 112±11 ml)and HCl (ALI: 119±12 ml; ALI + HS: 112±12 ml) were alsosimilar among the groups.

Hemodynamic and respiratory dataThe hemodynamic measurements were similar for each of

the investigated groups throughout the period of observa-tion, and significant changes were not observed in the MAP,CVP, or SVRI values. However, the pulmonary arterialpressure and PVRI values exhibited significant increasesafter T30 and remained high throughout the observationperiod in the ALI and ALI+HS groups (Table 1). FollowingHCl instillation, both lung injury groups exhibited significantreductions in static pulmonary compliance and increases inPplat (plateau pressure). The PaO2/FiO2 ratio exhibited asignificant reduction at T30 in injured animals, or those in theALI and ALI+HS groups (240±40 and 244±51, respec-tively, po0.001), compared with those in the control animals,or the Sham and HS groups (467±37 and 445±57,respectively), and this ratio remained low throughout theobservation period (Figure 2).

BAL cytokines, neutrophil count and oxidativeburst analysisALI animals showed higher TNF and IL-8 levels relative to

the Sham, HS and ALI+HS groups. Higher levels of IL-1B

were found in the ALI+HS group, followed by the ALI group(Figure 3).

The ALI and ALI+HS groups (34.4±3.5 and 30.2±7.7neutrophils x 103/ml, respectively) presented higher BALneutrophil counts relative to the Sham and HS groups(18.7±1.6 and 17.9±1.5 neutrophils x 103/ml, respectively).

Additionally, the ALI and ALI+HS groups presentedsignificantly higher activity (244.8±130 and 232.6±122GMFI, respectively) in BAL neutrophils in the PMA-inducedburst responses compared with that of the Sham and HSgroups (51.7±22 and 57±20.2 GMFI, respectively).

Histological analysisThe score for histological injury was significantly higher in

the ALI and ALI+HS groups compared with the Sham andHS groups (Figure 4).

The pattern of lung injury observed in the ALI and ALI+HS groups was heterogeneous and more evident in thediaphragmatic lobes. Examination of these lung tissuesrevealed large areas of alveolar architecture destruction,hemorrhage, edema and inflammatory polymorphonuclearand mononuclear cell infiltration (Figure 5). However,significant differences were not observed between the scoresexhibited by animals in the ALI and ALI+HS groups.

’ DISCUSSION

In the present study, we demonstrated that hypertonicsaline infused after intratracheal HCl instillation attenuatedincreases in BAL neutrophil counts and inflammatorycytokine concentrations. HCl instillation alone induced asevere direct lung injury as evidenced by an intenseinflammatory reaction observed in the lung histology, BALcytokine levels and oxidative burst. Lung function was alsoadversely affected, which was indicated by decreased gasexchange and reduced lung compliance.

Previous studies have attributed beneficial effects to hyper-tonic saline in a number of ALI models, such as oleic acid andischemia/reperfusion-induced lung injury (7,18,19). The use ofhypertonic saline solution has also demonstrated potential anti-inflammatory effects related to neutrophil activation (20) in cellcultures as well as in experimental models of sepsis andhemorrhagic shock (21-23). Hypertonic saline solution acts onpolymorphonuclear A2 adenosine receptors and causes afeedback mechanism that stimulates cAMP and PKA release,thus blocking neutrophil activation (21-23).

It is believed that hyperosmolar solutions can alsodecrease pulmonary vascular permeability and leukocyteadhesion molecule expression, especially P-selectin andL-selectin. This expression hinders neutrophil adhesion to theendothelium and may result in reduced lung injury (24,25).

Contrary to the results obtained in studies performed in aHCl-induced lung injury model (8) and an experimental oleicacid-induced lung injury model (7), which demonstrated thatpulmonary edema decreased in rats treated with 7.5%hypertonic saline, our histopathological results did not showa significant differences between the ALI and ALI+HSgroups with regard to the investigated parameters. However,the ALI+HS group tended to show lower histopathologicalscores relative to the ALI group, although this difference wasnot significant. Regarding the control groups, significantdifferences were not observed between the HS and Shamgroups, which demonstrates that the administration of 7.5%

Figure 2 - Changes in the PaO2/FiO2 ratio in the control (Shamand HS) and acid-lesion (acute lung injury and AI + hypertonicsaline) groups. *: po0.05 compared with TB; w: po0.05compared with the Sham group; #: po0.05 compared with thehypertonic saline group.

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hypertonic saline as an isolated agent did not result inworsening lung injury scores.Alveolar-capillary barrier destruction and increased micro-

vascular permeability are known to trigger the process oflung injury via acid aspiration, which leads to the activationof leukocytes and their migration to pulmonary tissue as wellas the production of numerous inflammatory mediators (26).

Therefore, our findings of alveolar and interstitial edema andpolymorphonuclear and mononuclear cells sequestration inresponse to injury are consistent with the data reported in theliterature.Although significant differences were not observed

between the ALI and ALI+HS groups, higher concentrationsof TNF-a and IL-8 in the ALI-group BAL were observed.

Figure 3 - Bronchoalveolar lavage cytokines. The values are expressed in pg/ml. Data are presented as the group mean±SD. w: po0.05compared with the Sham group; #: po0.05 compared with the hypertonic saline group; y: po0.05 compared with the acute lung injury group.

Figure 4 - Scores for histological injury of the lungs. w: po0.05 compared with the Sham group; #: po0.05 compared with thehypertonic saline group.

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The concentration of IL-10, an anti-inflammatory cytokine,was significantly lower in the ALI-HS group, which mayhave been caused by the lower TNF-a values in this groupbecause IL-10 expression is closely related to TNF-aexpression (27). However, BAL samples were collected270 min after acid administration, and the IL-10 concentra-tion may subsequently increase.The observed increases in MPAP and PVRI suggest that

pulmonary hypertension occurred as a result of the initiallung injury triggered by HCl. The two main causes werechemical injury, which led to the destruction of the alveolar-capillary barrier and an increase of pulmonary permeability(28), and pulmonary blood flow redistribution triggered by aphysiological process known as hypoxic pulmonary vaso-constriction (HPV), which optimizes the ventilation/perfu-sion ratio and blood oxygenation by gas exchange.Regarding the gas exchange, we also found a statistically

significant decrease in the PaO2/FiO2 ratio, which wasapproximately 50% lower in the ALI and ALI+HS groupscompared with the other groups. This finding is consistentwith the results obtained by Inci et al. (29) in rats after HClinstillation. The PaO2 values, however, were only signifi-cantly different among the injury and control groups at theT30 time point and returned to BL values at subsequent timepoints. Although the PaO2/FiO2 ratio gradually increasedthroughout the study, these values remained lower in thetreated groups compared with the non-injured groups.Although the PaO2 value and PaO2/FiO2 ratio gradually

increased over the observation period, a similar response was

not observed in pulmonary compliance, which remained lowerfor the duration of the study. Vascular occlusion and hypoxicvasoconstriction may shift blood from non-aerated to aeratedlung areas, thus contributing to oxygenation improvement.

In addition, to minimize secondary lung injury caused bythe ventilator, we limited the pressure-controlled ventilationto a volume of 8 ml/kg and 5 cmH2O PEEP. We alsomaintained Pplat values below 30 cmH2O throughout thestudy, which is similar to the standard defined for patientswith ARDS [30]. We observed a significant Pplat increase inanimals after HCl instillation, which was positively corre-lated with increases in the mPAP and PVRI values. More-over, the ALI and ALI+HS groups also showed significantlyreduced pulmonary compliance after HCl instillation com-pared with the control groups. These changes in ventilatoryparameters remained throughout the study period andcorroborate the results of other studies using HCl- or oleicacid-induced lung injury (29,31).

This ALI model was capable of inducing increasedchanges in ventilatory parameters, and it also induced therelease of inflammatory cytokines in the bronchoalveolarlavage samples in the ALI and ALI+HS groups. Thehistopathological analysis identified areas of heterogeneousinjury characterized by polymorphonuclear infiltrates, alveo-lar hemorrhage, and edema in the groups subjected to ALI.

Although the use of 7.5% hypertonic saline was shown tobe safe in our study, the benefits of its use remain uncertainbecause we did not observe improvements in any of theinvestigated parameters among animals in the injury group.

Figure 5 - Representative photomicrographs with hematoxylin and eosin (H&E) staining (x200) of the lungs of pigs submitted to acutelung injury. A) Sham group. B) hypertonic saline group. C) acute lung injury group. D) acute lung injury + hypertonic saline group.Extensive alveolar and interstitial inflammatory infiltration was seen in both injury groups.

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’ ACKNOWLEDGMENTS

This work was supported by grants from Fundacão de Amparo e Pesquisado Estado de São Paulo (FAPESP 08/55376-7 and 08/56732-4) and LIM08 (Medical Investigation Laboratories Institute, Hospital das Clinicas daFaculdade de Medicina).


Holms CA conducted the study and data analysis. Otsuki DA helped designthe study, conduct the study, collect and analyze data and prepare themanuscript. Kahvegian M helped conduct the study. Massoco CO helpedconduct the study and data analysis. Fantoni DT designed the study andhelped analyze the data. Gutierrez PS performed the histological analysis.Auler Jr JO helped design the study and prepare the manuscript. All of theauthors read and approved the final manuscript.

*Presented in part at the 30th International Symposium on Intensive Careand Emergency Medicine, 2010, Brussels.

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http://dx.doi.org/10.1007/s00134-007-0782-0

Operative versus nonoperative treatment for displacedmidshaft clavicle fractures: a meta-analysis based oncurrent evidenceXin-Hua Wang, Wei-Jun Guo, A-Bing Li, Guang-Jun Cheng, Tao Lei, You-Ming Zhao*

The Second Affiliated Hospital of Wenzhou Medical University, Department of Orthopedics Surgery, Wenzhou, China.

Literature searches of the Cochrane Library, PubMed, EMBASE, Web of Science, LILACS, China National KnowledgeInfrastructure, and Wanfang Data databases were performed from 1966 to September 2014. Only randomizedand quasi-randomized controlled clinical trials comparing operative and nonoperative treatments for displacedmidshaft clavicle fractures were included. Data collection and extraction, quality assessment, and data analyseswere performed according to the Cochrane standards. Thirteen studies were considered in the meta-analysis.Constant scores and the Disabilities of the Arm, Shoulder and Hand scores were improved in the operative fixationgroup at a follow up of one year or more. The nonunion and symptomatic malunion rates were significantlylower in the operative group. Additionally, the nonoperative group had a higher likelihood of neurologicalsymptoms compared with the operative group. A significantly higher risk of complications was found in patientstreated conservatively than in those who underwent operative fixation. However, when patients with nonunionand symptomatic malunion were excluded from the analysis, no significant differences in the complication ratewere found. We concluded that based on the current clinical reports, operative treatment is superior tononoperative treatment in the management of displaced midshaft clavicle fractures. However, we do not supportthe routine use of primary operative fixation for all displaced midshaft clavicle fractures in adults.

KEYWORDS: Clavicle; Midshaft clavicles; Operative treatment; Nonoperative treatment; Meta-analysis.

Wang XH, GuoWJ, Li AB, Cheng GJ, Lei T, Zhao YMOperative versus nonoperative treatment for displacedmidshaft clavicle fractures: a meta-analysisbased on current evidence. Clinics. 2015;70(8):584-592

Received for publication on February 04, 2015; First review completed on March 23, 2015; Accepted for publication on April 30, 2015



’ INTRODUCTION

Clavicle fractures, which account for approximately 2.6% oftotal body fractures and 34-45% of shoulder girdle injuries inadults, are among the most common bone injuries in the body(1,2). Appproximately 69-81% of clavicle fractures are in themiddle one-third of the clavicle, which is the thinnest part andcontains the smallest amount of soft tissue; 17% of claviclefractures are in the lateral one-third, and 2% are in the medialone-third (3). Conventionally, most acute displaced midshaftclavicles fractures are treated nonoperatively with the expecta-tionsof a high probability of fracture union, good functionaloutcomes and a high level of patient satisfaction (4-8).However, the outcomes of nonoperative treatment are not

as favorable as once thought, and the trend to surgically treatthese fractures has grown (9,10). Whether surgical treatmentis associated with improved outcomes remains unknown.

A few meta-analyses comparing operative versus non-operative approaches for the treatment of midshaft claviclefractures have been published in recent years (11,12), but theresults were inconclusive due to the relatively small samplesize in each published study. However, because severalrelevant studies have been published on this topic in recentyears, the present meta-analysis is more precise.

The purpose of the present systematic review and meta-analysis was to determine the effectiveness of operativeversus nonoperative treatment for displaced midshaftclavicle fractures by comparing the clinical results reportedin all of the available related evidence.


Literature searchThis study was performed with guidance from the

Cochrane Handbook for Systematic Reviews of Interventionsand the Preferred Reporting Items for Systematic Reviewsand Meta-Analyses statement (13,14). The PubMed,Cochrane Library, EMBASE, Web of Science, LILACS, ChinaNational Knowledge Infrastructure and Wanfang Datadatabases were searched (from 1966 to September 2014).Keywords combined with MeSH terms, including clavicle,DOI: 10.6061/clinics/2015(08)09


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clavicular, and fractures, were used for searching; the searchwas performed without language restrictions but waslimited to human subjects. Additionally, the reference listsof identified studies were manually checked to include otherpotentially eligible trials. This process was performediteratively until no additional articles could be identified.

Inclusion and exclusion criteriaThe search results were screened based on the following

inclusion criteria: (i) the studies were randomized or quasi-randomized controlled clinical trials on patients withdisplaced midshaft clavicle fractures that had occurredwithin less than two weeks; (ii) the studies comparedoperative (including plate, intramedullary nail fixation) withnonoperative treatment (including sling or a figure-eightbandage), (iii) the patients were at least 16 years of age; and(iv) the studies included comparisons of the functionaloutcomes, measured with Disabilities of the Arm, Shoulderand Hand (DASH) and Constant scores, nonunion, sympto-matic malunion, and complications. The exclusion criteriawere as follows: (i) studies including patients with patholo-gical fractures or preexisting shoulder abnormalities; (ii)studies concerning adolescent fractures; (iii) studies concern-ing open fractures; (iv) review literature, repeated reports,retrospective studies, or case reports; and (v) studies that didnot report outcomes of interest.

Data extractionTwo reviewers independently extracted the following data

from each included study: first author, year of publication,number of patients, number of patients lost to follow up,type of interventions, functional outcomes, and rates ofnonunion, symptomatic malunion, neurological symptomsand total complications.

Outcomes for analysisThe primary outcome was the incidence of nonunion and

symptomatic malunion; the secondary outcomes wereclinical function measured by the DASH and ConstantShoulder scores, complications and subgroup analyses(neurological symptoms and complications without non-union or symptomatic malunion).

Statistical analysisEstimates of the treatment effect were expressed as risk

ratios (RRs) for dichotomous outcomes and weighted meandifferences (WMDs) for continuous outcomes, both with 95%confidence intervals (CIs). For studies that did not presentstandard deviations, the standard deviations were calculatedfrom the P-value or CI following the guidance of theCochrane Handbook for Systematic Reviews of Interventions(13). Homogeneity across the studies was assessed with achi-square analysis, considering po0.10 significant. A fixedeffects model was used when the heterogeneity was notsignificant, and a random effects model was adopted ifsignificant heterogeneity was present. A sensitivity analysiswas performed by omitting one study each time to explorepotential sources of heterogeneity and to test the stability ofpooled results. Publication bias was observed with the funnelplot. Review Manager (RevMan) software (Version 5.3.5. TheNordic Cochrane Centre, Copenhagen, Denmark) (15),provided by The Cochrane Collaboration, was used forgraphical representation of the pooled data.

Critical AppraisalAll selected articles were critically appraised by two

reviewers independently, using the Jaded score (16). Thescore for each article could range from 0 (lowest quality) to 5(highest quality). Scores of 3 to 5 denote good to excellentquality, and scores of 0 to 2 denote poor to low quality. Alldisagreements between the authors were resolved byconsensus, and a third author was consulted if necessary.

’ RESULTS

Characteristics of the eligible studiesDetails of the literature search are presented in a flow

diagram (Figure 1). Thirteen studies with relatively lowquality were included in the final analysis. Among them, thereport by Smith et al. (17) was an abstract that met theinclusion criteria, and the sample sizes of the studies rangedfrom 40 to 178 patients. Information on the generalcharacteristics, participants, and methodological quality ofthe 13 studies is summarized in Table 1. Of a total of 959included patients, 507 were treated with operativeapproaches, and the others were treated with conservativeapproaches. Allocation concealment was reported in 8 trials(18-25) and was not stated in the other trials. Blinding wasrarely used in the included studies; only one study byRobinson et al. (23) was blinded in the functional assessment.

Nonunion and symptomatic malunionAll 13 studies reported nonunion incidences. The pooled

results of our primary outcome measure, nonunion incidence,presented a significant difference favoring operative overnonoperative treatment (RR, 0.16; 95%CI, 0.09-0.30;po0.00001). Subgroup analysis concerning fixation methodsshowed that plate fixation (RR, 0.15; 95%CI, 0.07-0.29;po0.00001) but not intramedullary nailing fixation (RR, 0.23;95% CI, 0.06-0.92; p=0.04) was associated with a reduced riskcompared with nonoperative treatment (Figure 2A). Onestudy reported by Böhme et al. (27), in which fractures werereduced and fixed with both plates and nails in the operativegroups, was excluded from the subgroup analysis.Information on the incidence of symptomatic malunion

was provided in 9 studies (17,18,20-22,24-27). Using the fixedeffects model, the rate of symptomatic malunion wassignificantly lower in the operative group compared withthat in the nonoperative group (RR 0.13, 95%CI 0.07–0.24,po0.00001) (Figure 2B). No significant heterogeneity wasdetected among these studies (Chi2=10.89, df=12, I2=0%,p=0.74 and Chi2=2.46, df=8, I2=0%, p=0.96, respectively).

Functional outcomesNine studies (18,10-23,25,27-29) reported Constant scores

(eight at a follow up of one year or more and one at a followup of 6 months); the Constant scores of the operative groupwere higher than those of the nonoperative group. Three(27-29) of the nine studies were excluded from the analysisdue to a lack of data on the standard deviation or toinsufficient follow-up. The test for heterogeneity wassignificant (Chi2=14.13, df=5, I2=65%, p=0.01). Using therandom effects model, the aggregated results suggested thatthe Constant score was significantly higher in the operativegroup compared with the nonoperative group (WMD 4.74,95%CI, 2.45–7.03, po0.0001) (Figure 3A). Subsequently, weperformed a sensitivity analysis to explore potential sourcesof heterogeneity. Exclusion of the trial conducted by

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CLINICS 2015;70(8):584-592 A meta-analysis of intervention for clavicleWang XH et al.

Mirzatolooei (21) reduced the heterogeneity (Chi2=2.47, df=4,I2=0%, p=0.65) but did not materially alter the pooled results(WMD 3.74, 95%CI, 2.39 – 5.08, po0.00001).

DASH scores were used in eight studies (18, 20-23,25,28,29);the DASH scores of the operative group were lower than thoseof the nonoperative group at a follow up of one yearor more, but the actual standard deviations were only includedin five studies (18,21-23,25). Pooled data showed that theDASH score in the operative group was significantly lowerthan that in the nonoperative group (WMD -6.34,95%CI -11.28 – -1.39, p=0.01) (Figure 3B). Significant hetero-geneity was detected among these studies (Chi2=33.93, df=4,I2=88%, po0.00001). Similarly, exclusion of the trial con-ducted by Mirzatolooei (21) resolved the heterogeneity(Chi2=2.77, df=3, I2=0%, p=0.43) without materially alteringthe pooled results (WMD -3.64, 95%CI -5.58 – -1.69,p=0.0002).

ComplicationsBecause the definition of complications varied in all of the

studies, we defined complications as all adverse events thatwere reported in those trials: nonunion (usually defined as noevidence of healing at fifty-two weeks after injury), delayedunion (no evidence of healing at twenty-four weeks afterinjury), symptomatic malunion, infection, hardware removal,neurological symptoms, and refracture, among others.

In an overall analysis of the 13 selected studies, signifi-cant heterogeneity (Chi2=22.50, df=12, I2=47%, p=0.03) wasdetected among these studies. Sensitivity analysis found thatthe study reported by Judd et al. (19) was the source ofheterogeneity, probably owing to a high rate of hardware-related complications associated with the use of Hagie pinsin this study. Thus, the random effects model was applied. Asignificantly higher risk of complications was found inpatients treated conservatively than in those who underwent

Figure 1 - Flow chart showing article selection.

Table 1 - Characteristics and methodological quality of the included studies.

Study Design No. of Patients Assessed(O/N)

Range of Ages(years)

Follow-up(months)

InternalFixation

NonoperativeTreatment

JadadScore

Smith (2001) RCT 30/35 Adults 12 Plate Sling 3Jubel (2005) QRCT 26/27 Adults 6 Nail Bandage 1COTS (2007) RCT 62/49 16–60 12 Plate Sling 3Figueiredo (2008) RCT 24/16 18-58 12 Plate Sling 3Judd (2009) RCT 29/28 17–40 12 Nail Sling 3Smekal (2009) RCT 30/30 18–65 24 Nail Sling 3Bohme (2011) QRCT 58/38 18-70 8 Plate/Nail Bandage 1Chen (2011) RCT 30/30 18-63 15 Nail Sling 3Mirzatolooei (2011) RCT 26/24 18–65 12 Plate Sling 3Kulshrestha (2011) QRCT 45/28 20-50 18 Plate Sling 1Virtanen (2012) RCT 26/25 18–70 12 Plate Sling 3Robinson (2013) RCT 86/92 16–60 12 Plate CollarCuff 4Mohsen (2014) QRCT 35/30 18-60 6 Plate Bandage 1

O/N: operative group/nonoperative group, RCT: randomized controlled trial, QRCT: quasi-randomized controlled trial.

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CLINICS 2015;70(8):584-592

operative fixation (RR 0.57, 95%CI 0.46-0.72, po0.00001)(Figure 4A). Furthermore, we also performed a subgroupanalysis of the complications without nonunion and sympto-matic malunion. The aggregated results suggested that therewere no significant differences between groups in the rates ofcomplications (RR 1.30, 95%CI 0.88–1.92, p=0.19) (Figure 4B).Significant heterogeneity was detected among these studies(Chi2=25.81, df=12, I2=54%, p=0.01). We then performedsensitivity analysis and found that the study reported byMohsen et al. (29) was source of heterogeneity (Chi2=12.95,df=11, I2=15%, p=0.30). Although heterogeneity was found,statistically similar results to those of the overall analysiswere obtained in the sensitivity analysis. The predominant

complications in the nonoperative group were nonunion,neurological symptoms (including brachial plexus irritationand compression) and symptomatic malunion. The operativecomplications tended to be hardware related (including plateirritation, pin protrusion and removal).

Neurologic symptomsNine studies reported neurological symptoms (17-22,25,

27,28). Pooled data showed that the operative group had asignificantly lower likelihood of developing neurologicalsymptoms compared with the nonoperative group (RR 0.40,95%CI 0.23–0.70, p=0.001). No significant heterogeneity was

Figure 2 - Forest plot showing comparison of nonunion rate (A) and symptomatic malunion rate (B) between operative (experimental)and nonoperative (control) groups.

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detected among these studies (Chi2=8.07, df=8, I2=1%, p=0.43)(Figure 4C).

Publication biasPublication bias was assessed by comparing the WMDs of

nonunion; no evidence of publication bias was detected(Figure 5).

’ DISCUSSION

Our meta-analysis revealed that primary operative fixationcould effectively reduce the rates of nonunion, symptomaticmalunion, neurological symptoms and overall complica-tions. In addition, DASH and Constant scores weresignificantly improved after operative fixation comparedwith nonoperative treatment after a follow up of one year ormore. Based on current clinical reports, we conclude thatoperative treatment is superior to nonoperative treatment inthe management of displaced midshaft clavicle fractures.Pooled data showed that 14% of 452 patients in the

nonoperative group developed a nonunion, which issignificantly higher (p=0.00001) than the 1.7% rate ofnonunion in the 507 patients of the operative group.Symptomatic malunion was also significantly more commonin the nonoperative group (20% in the nonoperative groupversus 1.8% in the operative group, po0.00001). However,with the data available, we were not able to draw any

specific conclusions as to which patients were most likely tosuffer from one of these significant complications; we also donot support the routine use of primary operative fixation forall displaced clavicle fractures in adults because an unac-ceptably high number of patients would be exposed to therisks of surgery (23).

Although significant differences were found between thetwo treatment groups in terms of functional outcomes, thereasons for functional outcome differences are probablymultifactorial because most individuals who developednonunion or symptomatic malunion had significantly loweroutcome scores (i.e., a mean of sixteen points worse on theDASH score in nonunion patients in the study by Virtanenet al. (22)). Additionally, Robinson et al. (23) showed that thedevelopment of nonunion was the only independentpredictor of functional outcomes. When patients withnonunions were excluded, there was only a trend towardbetter functional outcomes in the operative group, with nosignificantly different scores at any time. Therefore, wethought that the improved outcomes may have resulted fromthe prevention of nonunion and symptomatic malunion byoperative fixation. Unfortunately, without sufficient originaldata, we cannot perform a subgroup analysis of patientswith overall healed fractures.

Overall complication and neurological symptom rateswere higher in the nonoperative group than in the operativegroup. The subgroup analysis of the complications without

Figure 3 - Forest plot showing comparison of Constant scores (A) and DASH scores (B) between operative (experimental) andnonoperative (control) groups.

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CLINICS 2015;70(8):584-592

nonunion and symptomatic malunion suggested that nosignificant between-group differences existed in the com-plication rates (p=0.23). The most common complications inthe operative group were hardware related (including plateirritation, pin protrusion and removal). Theoretically, thesecomplications could be reduced by using less prominent

implants or improved surgical techniques. The predomi-nant complications in the nonoperative group were non-union, neurological symptoms (including brachial plexusirritation and compression) and symptomatic malunion;however, most of those complications require operativeintervention.

Figure 4 - Forest plot showing comparison of complications rates (A and B) and neurologic symptoms rates (C) between operative(experimental) and nonoperative (control) groups.

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Although modern plate fixation techniques provide reli-able healing rates, the optimal plate position and type remaincontroversial. The clavicle contour and anatomy are curvedin multiple planes. The reconstruction plate is easier tocontour in all planes than the stiffer dynamic compressionplates (DCP), which allow bending only along the length ofthe plate. For superior plating, a reconstruction plate orprecontoured plate can more precisely fit the ‘S’-shapedanatomy. For anteroinferior plating, DCPs can be bent toconform to the anatomy very well (39). Regarding stability,two biomechanical studies have found greater stability withcompression plates compared with reconstruction plates(31,32). In addition, a finite element study showed thatanteroinferior plating best resists the effects of most dailyliving forces that act on the clavicle and can be consideredmore mechanically physiological (33).Will et al. (34) suggested that locked compression plates

(LCPs) provided more stiffness and less deflection than low-contact dynamic compression plates (LC-DCPs). Using asimulated segmental clavicle fracture model, another bio-mechanical study by Iannotti et al. (35) reported that LC-DCPs offer significantly greater biomechanical stability thanreconstruction plates and DCPs and that clavicles plated atthe superior aspect exhibited significantly greater biomecha-nical stability than those plated at the anterior aspect.However, most of the biomechanical studies must beinterpreted with caution because such testing can offer cleancomparisons of instrumentation and technique withoutthe confounding factors of patient and surgeon variations.Nine of the 13 studies included in this review used platefixation; among them, three studies used reconstruction

plates (21,22,26), two studies used DCPs (24,29), and twostudies used LCPs (17,23). Another two studies that used amixture of plate implants (18,27) was not analyzed here. Therate of nonunion was 6% in the DCP group, which washigher than that in the reconstruction plate group (1%) andLCP group (1%). Symptomatic malunion only occurred in thereconstruction plate group, with a rate of 6%. In addition,overall complication rates were higher than 20% in all of thegroups, which is consistent with previous literature (30). Recentclinical studies have shown efficient healing, few complications,and excellent return to function for anteroinferior plating(36-38). The advantages of this technique include the avoidanceof potentially dangerous infraclavicular structures and thereduction of patient complaints due to implant prominence (36).A retrospective cohort study (39) with 156 midshaft claviclefractures concluded that anteroinferior clavicle fracture fixationwith DCPs results in excellent healing rates and lower removalrates. Moreover, implant failure occurred more often withreconstruction plates compared with dynamic compressionplates (p=0.029).

Considering these mechanical and clinical findings, theplate type, precontouring, and position likely affect out-comes and implant-related complication rates. However,these effects have yet to be fully examined and moreprospective trials are required to analyze the influence ofvarious plate types and positions on implant-related com-plications in the future.

Further subgroup analysis by type of surgery indicatedthat plate fixation but not intramedullary nail fixationwas associated with a reduced risk of nonunion, sympto-matic malunion and total complications compared with

Figure 5 - Funnel plot of detection of publication bias.

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nonoperative treatment. No significant difference in thefunctional outcomes between these two techniques wasobserved. However, the pooled data showed that theincidences of nonunion, symptomatic malunion and totalcomplications were comparable in the two operative groups(2% versus 1%, 2% versus 0, 27% versus 27%, respectively).Both the plating and intramedullary nail methods have

advantages and disadvantages. Biomechanically, plate fixa-tion is superior to intramedullary fixation (40). Patientstreated with plate fixation can achieve full range of motion.The disadvantages of plate fixation include the necessity forincreased exposure and soft tissue stripping, the increasedrisk of damage to the supraclavicular nerve, slightly higherinfection rates, and the risk of refracture after plate removal(18). A recent randomized clinical trial comparing lockedintramedullary nailing versus plating for displaced midshaftclavicle fractures performed by Ferran et al. (41) showed nosignificant differences between the two operative techniques.Another two prospective comparative studies concluded thatboth plating and intramedullary flexible nailing are equallyeffective alternatives for the surgical fixation of displacedmidshaft clavicle fractures but that intramedullary techni-ques have potential advantages such as less soft tissue injury,shorter operative times and hospital stays, less blood lossand higher cosmetic satisfaction (42,43). The subgroupanalysis results of our review are partially consistent withthe results of the three trials; however, due to the limitationsof those studies such as their small sample sizes and singlecenter designs, we cannot provide any strong conclusions.Studies with sound rationale and design are required toaccurately and definitively assess the differences in outcomesbetween plate fixation and intramedullary fixation (44).However, with a myriad of options available for both plate

fixation and intramedullary fixation, the question of whichform of fixation is superior remains. According to currentlyavailable data, the superior surgical technique and implantchoice are those that the surgeon was originally trained toperform and use.Previous literature has addressed the issue of nails, yet the

difference between locked and unlocked nails has not beenconsidered. This study may provide additional interestingclues for future research on this topic.We identified six systematic reviews that approached the

comparison between surgical versus conservative interventionsto treat middle-third clavicle fracture in adults (11,12,45-48).The results of our review are consistent with those of the eightsystematic reviews; however, the conclusions of those pub-lished reviews varied, which was partly in accordance with ourconclusions. The only Cochrane systematic review, reported byLenza et al, used more comprehensive statistical methods,which were lacking in our present study. The most distinguish-ing characteristic of those published reviews was that analysiswas conducted with incomplete information. With the excep-tion of one study by Lenza et al, the reviews did not include thetwo RCTs (24,25). Additionally, new RCTs have been publishedsince then. Our review adds consistent information for currentclinical practice. We applied more specific subgroup analysis bysurgery and implant type and then discussed the results indetail. Furthermore, we performed a broader literature searchthat included non-English publications. Bias is inherent inmany analyses focusing on specific populations or geographicareas. By including all of the available studies, including thosefrommultiple countries and reported in multiple languages, webelieve that our conclusions are applicable to most populations.

There are some limitations of this study. First, the recruitedstudies were not all randomized controlled trials. The lack ofinadequate allocation concealment and blinding, which canlead to over-reporting of the treatment effect and selection orallocation biases, likely affected the study results. Second, thepreoperative fracture pattern was found to be significantlyrelated to implant failure (49), but our meta-analysis couldnot show fracture type–specific effects between operativeand nonoperative treatments because of the limited data ofthe studies. Finally, despite our best efforts to use multiplesearch methods, we may not have detected all eligibleexisting trials with results that are applicable to our meta-analysis. Therefore, the results should be interpreted withcaution. Further research entailing high-quality randomizedcontrolled, multicenter trials with long-term functional out-comes and fracture type–specific designs is required to addresskey clinical questions regarding the effects of operativetreatment versus nonoperative treatment in the managementof displaced midshaft clavicle fractures in adults.In summary, we conclude that operative treatment is

superior to nonoperative treatment in the management ofdisplaced midshaft clavicle fractures, although we do notsupport the routine use of primary operative fixation for alldisplaced clavicle fractures in adults. Patients with acompletely displaced midshaft clavicle fracture should beinformed that they will be at a higher risk of sustainingnonunion, symptomatic malunion and potential neurologicalsymptoms if the fracture is treated conservatively.


Wang XH conceived the study, collected the data, participated in the analysisof samples, drafted the manuscript and performed the statistical analysis. GuoWJ conceived the study and participated in its design, coordination anddrafting. Li AB participated in the analysis and interpretation of samples andin the language translation of non-English studies. Cheng GJ participated inthe language translation of non-English studies. Lei T participated in therevision of the manuscript. Zhao YM participated in the review, revision,coordination and drafting of the manuscript and performed the analysis withconstructive discussions. The aim of this article was to identify the effects ofoperative versus nonoperative treatment in the management of displacedmidshaft clavicle fractures in adults.

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The association between the rs11196218A/G poly-morphism of the TCF7L2 gene and type 2 diabetes inthe Chinese Han population: a meta-analysisEnting Ma,I Huili Wang,II Jing Guo,I Ruirui Tian,III Li WeiIV,*

IGeneral Hospital of Tianjin Medical University, Department of Pediatric Ward, Tianjin, China. IIXi’an International University, Department of Nursing,

Xi’an, China. IIITianjin Medical University, Department of Nursing, Tianjin, China. IVGeneral Hospital of Tianjin Medical University, Department of Surgery,

Tianjin, China.

Transcription factor 7-like 2 has been shown to be associated with type 2 diabetes mellitus in multiple ethnic groupsin recent years. In the Chinese Han population in particular, numerous studies have evaluated the associationbetween the rs11196218A/G polymorphism of the transcription factor 7-like 2 gene and type 2 diabetes mellitus.However, the results have been inconsistent, so we performed a meta-analysis to assess the association. Odds ratioand 95% confidence interval values were calculated using a random-effects model or a fixed-effects model based onheterogeneity analysis. The quality of the included studies was evaluated using the Newcastle-Ottawa Scale.Subgroup analyses were conducted based on conformity with Hardy-Weinberg equilibrium in the control group aswell as on other variables, such as age, sex and body mass index. Sensitivity analysis was also performed to detectheterogeneity and to assess the stability of the results. In total, 10 case-control studies comprising 7,491 cases and12,968 controls were included in this meta-analysis. The combined analysis indicated that the rs11196218A/Gpolymorphism was not associated with type 2 diabetes mellitus (G vs. A, OR=1.04, 95% CI=0.97-1.13, p=0.28). Thesubgroup analyses also did not show any association between the rs11196218A/G polymorphism and the risk of type2 diabetes mellitus. Furthermore, the results of the subgroup analyses indicated that the absence of an associationwas not influenced by age, sex or body mass index. The results of the sensitivity analysis verified the reliability andstability of this meta-analysis. In conclusion, this study indicated that there is no significant association between thers11196218A/G polymorphism and the risk of type 2 diabetes mellitus in the Chinese Han population.

KEYWORDS: Type 2 diabetes mellitus (T2DM); Transcription factor 7-like 2 (TCF7L2); rs11196218A/G polymorphism;Meta-analysis.

Ma E, Wang H, Guo J, Tian R, Wei L. The association between the rs11196218A/G polymorphism of the TCF7L2 gene and type 2 diabetes in theChinese Han population: a meta-analysis. Clinics. 2015;70(8):593-599

Received for publication on January 29, 2015; First review completed on March 20, 2015; Accepted for publication on May 12, 2015



’ INTRODUCTION

Type 2 diabetes mellitus (T2DM) is a complex metabolicdisease resulting from a combination of environmental andgenetic factors (1). With the development of society and theimprovement of people’s standard of living, the prevalenceof T2DM is increasing rapidly all around the world. T2DM isvery harmful to human health and life, and it also confers aheavy burden on society.The transcription factor 7-like 2 (TCF7L2) gene, located on

chromosome 10q25 (2), is part of the Wnt signaling pathway (3)and has been shown to be strongly associated with an increased

risk of T2DM in Icelandic (4), European (5-11), West African (12),southern Asian (13), eastern Asian (14), and Chinese (15-19)populations. The TCF7L2 gene is regarded as one of the mostimportant genes for determining genetic susceptibility to T2DMthat has been identified in humans so far.With the growth of research efforts, studies on the association

between this variation and T2DM have been extensivelyperformed in China, but the results are disputable. Overall,the most significant risk locus identified in Chinese individualsis rs11196218. Therefore, only the rs11196218A/G polymorph-ism was considered in the present meta-analysis.In contrast to a single study, meta-analyses are based on all

available studies, which has improved the statistical powerfor exploring the above associations and has led to morereliable conclusions in recent years (20). Therefore, in thepresent study, a meta-analysis was performed to allow avaluable conclusion to be drawn regarding the relationshipbetween the rs11196218A/G polymorphism and T2DM riskin the Chinese Han population.DOI: 10.6061/clinics/2015(08)10


593

REVIEW


Our study adhered to the PRISMA Statement regardingthe reporting of systematic reviews and meta-analyses (21).

Search strategyIn this meta-analysis, we searched articles in PubMed,

Elsevier, SpringerLink, Embase, the Cochrane Library, ISIWeb of Science, Google Scholar and the China NationalKnowledge Infrastructure (CNKI). The search languagesincluded English and Chinese. The following key words andsubject terms were used: TCF7L2, transcription factor 7-like2, rs11196218, diabetes mellitus, type 2, type 2 diabetesmellitus, T2DM, and T2D. The reference lists of eligiblestudies and relevant review papers were additionallyidentified via a manual search on this topic. The last researchupdate was performed on July 15, 2014.

Inclusion and exclusion criteriaThe primary studies included in our meta-analysis had to

meet the following criteria: (1) the association betweenthe TCF7L2 polymorphism (rs11196218) and T2DM risk inthe Chinese Han population was clearly evaluated, (2) thediagnosis of T2DM and the sources of the cases and controlswere clearly described, (3) a case-control study design wasemployed, and (4) original data and sufficient informationwere provided to estimate the odds ratio (OR) and thecorresponding 95% confidence interval (95% CI). The majorreasons for exclusion were as follows: (1) duplicate data werepresented; (2) the article consisted of an abstract, comment, orreview or focused on pathological mechanisms; and (3) morethan one article was published by the same author using thesame data series, in which case the most recent publishedpaper or the paper with the largest sample size was selected.

Quality assessmentThe Newcastle-Ottawa Scale (NOS) (22) was used to assess

the quality of the studies included in our meta-analysis. TheNOS contains eight items and is categorized into threedimensions: selection, comparability and exposure, forcase-control studies. In particular, the selection dimensioncontains four items, the comparability dimension containsone item, and the exposure dimension contains three items.A star system is used to allow semi-quantitative assessmentof study quality, and a study can be awarded a maximum ofone star for each numbered item within the selection andexposure categories. Meanwhile, a maximum of two starscan be given for comparability. The NOS ranges from zero upto nine stars, as follows: high-quality study: more than sevenstars; medium-quality study: between four and six stars;poor-quality study: less than four stars.

Data extractionFor quality control, the data were extracted by two

reviewers using a standardized extraction form. If theinformation on the genotype distribution was inadequate,we tried to contact the authors by telephone or e-mail. Thefollowing information was extracted from each article:the last name of the first author, the year of publication,the region, the numbers of cases and controls, the source ofthe controls, the numbers of genotypes for cases andcontrols, matching factors, and the Hardy-Weinberg equili-brium (HWE) in each control group. Disagreement wasresolved by consulting a third reviewer.

Ethics statementThis article reports a meta-analysis of data obtained from

previous studies. All of the data were anonymized beforeanalysis. We also confirmed that none of the data involvedcompeting interests.

Statistical analysisIn this meta-analysis, OR and 95% CI values were used to

assess the association between a polymorphism of theTCF7L2 gene (rs11196218: G vs. A) and T2DM risk. Inaddition, statistical significance was assessed using a Z-test,and po0.05 indicated statistical significance for the associa-tion. The model selection was based on the heterogeneitytest; therefore, the w2-based Q-test was performed in thisstudy (23). When the Q-test yielded a p-value of morethan 0.10, a fixed-effects model was used (24); otherwise,a random-effects model was applied (25). Heterogeneitywas also assessed using the I2 test. The I2 statistic wasspecifically documented for the percentage of studyvariability observed due to heterogeneity rather thanchance (I2= 0-25%, no heterogeneity; I2= 25-50%, moderateheterogeneity; I2= 50-75%, high heterogeneity; I2= 75-100%,extreme heterogeneity) (26).

Subgroup analyses were performed based on HWE.Specifically, the p-values for HWE in the control group weredetermined using the Pearson chi-square test, regardless ofwhether the authors provided these values. HWE is theprincipal law of population genetic studies: if p40.05,the focus conforms to HWE, and the control samples arerepresentative. In Europeans, when excessive caloriesare consumed, variation in TCF7L2 can cause impairedb-cell function, indicating that body mass index (BMI) caninfluence the effect size of TCF7L2 variants; hence, these two

Figure 1 - Selection of the included studies.

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CLINICS 2015;70(8):593-599

factors will ultimately affect each other (27-29). Thisinteraction may also exist in Asians. For this reason, weconsidered whether age, sex and BMI were matched betweencases and controls in our subgroup analyses. For example,the studies showing a significant difference in age (po0.05)between the cases and the controls were assigned to theincomparability subgroup of age, whereas those exhibitingp40.05 were assigned to the comparability subgroup.However, due to the limited data in the studies, subgroupanalyses based on environment were not conducted.Sensitivity analysis was also performed to search for

heterogeneity and to assess the stability of the results. Onecase-control study was omitted each time to reflect theinfluence of each dataset on the pooled OR.Additionally, funnel plots were used to evaluate publica-

tion bias. All p-values were two tailed. Review Manager 5.0(2011, The Cochrane Collaboration) software was used toperform the meta-analysis.

’ RESULTS

Studies and data included in this meta-analysisIn total, 295 articles were relevant to our search terms, of

which 285 papers were excluded. Thus, 10 case-controlstudies (15-19,30-34) comprising 7,491 cases of T2DM and12,968 controls were ultimately included in this meta-analysis (Figure 1). All of these studies were published from2007-2014.The characteristics of these studies are summarized in

Table 1. The genotype frequency of the single nucleotidepolymorphism (SNP) was consistent with HWE in thecontrol group (p40.05) in all studies except for two (32,33).The quality assessment of all included studies, evaluatedaccording to the NOS, is provided in Table 2. Most studieswere of high quality in terms of selection and exposure.However, the quality of comparability was relativity low, asonly 3 studies (15,33,34) were comparable with the controlsregarding age, sex and BMI.

Association between rs11196218A/G polymorphismand T2DM riskUsing the heterogeneity test, we detected heterogeneity

among the studies in comparisons of the risk of carrying

allele G. Thus, a random-effects model was applied for themeta-analysis. The results indicated that the rs11196218A/Gpolymorphism was not associated with the risk of T2DM(G vs. A: OR=1.04, 95% CI=0.97-1.13, p=0.28; heterogeneitytest w

2= 17.71, p = 0.04, I2=49%; Figure 2).

The subgroup meta-analysis of the studies that exhibitedconsistency with HWE in the control group also showed thatthere was no association between the rs11196218A/Gpolymorphism and T2DM risk (G vs. A: OR= 1.08, 95%CI = 0.98-1.19, p = 0.12; heterogeneity test w

2= 15.79, p= 0.03,

I2=56%; Figure 3).The subgroup meta-analysis of the studies based on age

indicated that there was no significant association betweenthe rs11196218A/G polymorphism and T2DM risk (sub-group of age comparability: OR= 1.11, 95% CI = 0.96-1.28,p= 0.18; heterogeneity test w

2= 14.81, p = 0.01, I2= 66%; sub-

group of age incomparability: OR= 1.00, 95% CI = 0.94-1.06,p=0.93; heterogeneity test w

2= 1.45, p = 0.69, I2=0%).

The subgroup meta-analysis of the studies based on sexdemonstrated that there was no significant associationbetween the rs11196218A/G polymorphism and T2DMrisk (subgroup of sex comparability: OR = 1.10, 95% CI =0.98-1.24, p = 0.12; heterogeneity test w

2= 13.04, p = 0.02,

I2 = 62%; subgroup of sex incomparability: OR= 0.98, 95%CI = 0.91-1.05, p = 0.49; heterogeneity test w

2= 1.58, p = 0.66,

I2 = 0%).Finally, the subgroup meta-analysis of the studies based on

BMI illustrated that there was no significant associationbetween the polymorphism and T2DM risk (subgroup of

Table 1 - The basic characteristics of the included studies addressing rs11196218.

First

author

Publication

year

Region Source of

controls

Sample sizes Cases Controls HWE Comparability

Case Control G A G A

Ng 2007 Hong Kong Community-based andhospital staff

433 419 676 190 599 239 Yes Age*, sex*, BMI**

Zhang 2008 Hunan Hospital-based 536 475 716 272 623 221 Yes Age*, sex**, BMI*Luo 2009 Beijing Hospital-based 500 500 684 252 696 240 No Age**, sex**, BMI**Ma 2010 Shanghai Hospital-based 259 200 309 209 240 168 Yes Age**, sex*, BMI**Lin 2010 Chengdu Hospital-based 1529 1439 2171 887 2015 863 Yes Age**, sex*, BMI**Wen 2010 Shanghai N/D 1165 1136 1699 629 1677 595 No Age*, sex*, BMI*Zhu 2011 Anhui Hospital-based 300 300 156 444 122 478 Yes Age*, sex*, BMI*Zheng 2012 Chongqing Hospital-based 227 152 340 114 218 86 Yes Age*, sex**, BMI**Qiao 2012 Harbin Hospital-based 700 560 1025 367 819 295 Yes Age*, sex*, BMI*Zhai 2014 Henan Community-based

and Hospital-based1842 7777 2639 967 111287 4041 Yes Age**, sex**, BMI**

Abbreviations: HWE, Hardy-Weinberg equilibrium; Yes, the genotype distribution conformed to HWE in the control group; No, the genotype distributionnot conform to HWE in the control group;Hospital-based: subjects who were enrolled from health checks conducted at the hospital; community-based and hospital staff: subjects who wereenrolled from the community and hospital staff; N/D: no description; *: p40.05; **: po0.05.

Table 2 - Quality assessment of all of the included studies.

First author Publication year Selection Comparability Exposure

Ng 2007 $$$ $ $

Zhang 2008 $$ $ $$

Luo 2009 $$ $ $$

Ma 2010 $$ $ $$

Wen 2010 $$ $$ $$$

Lin 2010 $$ $ $$

Zhu 2011 $$$$ $$ $$

Zheng 2012 $$$$ $ $$

Qiao 2012 $$$ $$ $

Zhai 2014 $$ $ $$

595

CLINICS 2015;70(8):593-599 A meta-analysisMa E et al.

BMI comparability: OR= 1.03, 95% CI = 0.90-1.17, p= 0.72;heterogeneity test w

2= 6.21, p = 0.10, I2= 52%; subgroup of

BMI incomparability: OR= 1.06, 95% CI = 0.96-1.18, p= 0.26;heterogeneity test w

2= 11.32, p= 0.05, I2= 56%).

Sensitivity analysisA single study included in the meta-analysis was omitted

each time to reflect the influence of each dataset on thepooled OR values. We found that no single study couldchange the pooled results (Table 3), which indicated that theresults were relatively reliable.

Publication biasThe shape of the funnel plots was symmetrical, suggesting

that there was no evidence of publication bias for thers11196218A/G polymorphism (Figure 4).

’ DISCUSSION

Since the initial discovery that TCF7L2 is strongly associatedwith an increased risk of T2DM in Icelandic populations in2006 (4), many replication studies have confirmed the role ofTCF7L2 in conferring susceptibility to T2DM in differentpopulations and ethnic groups (5,7,11,14,38,39), especially forthe rs7903146C/T polymorphism. However, in the Chinese

population, rs7903146 is very rare, and several studies haveindicated that rs11196218 is the most significant risk variant(17). Nevertheless, the results regarding the rs11196218A/Gpolymorphism and T2DM risk have been inconsistent for theChinese Han population. Certain studies (15-19) confirmed thatthe G allele of the rs11196218A/G polymorphism wassignificantly associated with T2DM risk, whereas studies byMa (30), Zheng et al. (31), Luo et al. (32), Wen et al. (33) andQiao et al. (34) did not find a significant association betweenthe rs11196218A/G polymorphism and T2DM risk in theChinese Han population. Therefore, to resolve the conflictamong these studies, we performed a meta-analysis to assessthe association between the rs11196218A/G polymorphismand T2DM risk.

Previously, Luo et al. (32) conducted a meta-analysis toevaluate the effect of TCF7L2 on genetic susceptibility toT2DM in the East Asian population. However, only onearticle analyzed included samples from the Chinese Hanpopulation. In the current study, to increase the statisticalpower of the analysis, a larger amount of data was collectedfrom the literature to perform an up-to-date meta-analysisfor the Chinese Han population.

Our meta-analysis, which included 7,491 T2DM cases and12,968 controls from 10 case-control studies, explored theassociation between rs11196218A/G and T2DM risk. Overall,

Figure 2 - Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (G vs. A). n indicates the totalnumber of G alleles, and N indicates the total number of G alleles plus A alleles.

Figure 3 -Meta-analysis of the association between the rs11196218A/G polymorphism and T2DM risk (subgroup analyses for HWE in thecontrol group: G vs. A). n indicates the total number of G alleles, and N indicates the total number of G alleles plus A alleles.

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CLINICS 2015;70(8):593-599

we did not find that the rs11196218A/G polymorphism wassignificantly associated with an increased risk of T2DM inthe Chinese Han population, which is consistent with thefindings of Ma (30), Zheng et al. (31), Luo et al. (32), Wen etal.(33) and Qiao et al. (34). However, a previous meta-analysis performed by Luo et al. (32) found that rs11196218showed a marginal association with T2DM risk (OR= 1.09,95% CI = 1.00-1.19, p= 0.059). The most plausible explanationfor this discrepancy is the different genetic backgroundsassociated with different ethnic groups, areas and populationsubstructures.In the present study, obvious heterogeneity was observed

in the comparisons among studies. Thus, a random-effectsmodel was applied for the meta-analysis, after which weconducted a sensitivity analysis to identify the source of theheterogeneity. After omitting the study by Ng et al. (17) fromthe analysis, there was no heterogeneity among theremaining studies. One plausible explanation for theinfluential role of this study was that the populationsexamined by Ng et al. (17) resided in the metropolis of HongKong; as the residents of Hong Kong emigrated fromdifferent areas of China, they have probably undergonepopulation admixture. Moreover, T2DM is a complexhereditary disease, so the historical immigration, complexancestries, population movement, and recent intermarriages

with other ethnic groups that have occurred in Hong Kongmay have resulted in different risk factors in different people(40). Therefore, the genetic origins of risk may be relativelydifferent between this region and other areas. Pritchard et al.(41) also suggested that low diversity may be sufficient tolead to different results. However, although heterogeneitywas observed among studies, the results of the sensitivityanalysis verified the reliability and stability of the presentmeta-analysis.T2DM is a complex hereditary disease caused by

genetic and environmental factors (1). Other confound-ing factors, such as age, sex, BMI, the environmentand sophisticated gene-gene and gene-environmentinteractions, may also affect the results of studies onT2DM. Thus, the subgroup analyses in the present studywere also conducted based on variables such as age, sexand BMI. However, the results indicated that theassociation of the rs11196218A/G polymorphism withT2DM was not influenced by age, sex or BMI in thestudied population. Due to the limited data available inthe studies analyzed, subgroup analyses based on theenvironment and gene-gene and gene-environment inter-actions were not conducted.There were certain advantages of our meta-analysis.

First, to the best of our knowledge, this is the mostcomprehensive meta-analysis of the association betweenthe rs11196218A/G polymorphism of the TCF7L2 geneand T2DM risk in the Chinese Han population conductedto date, and our analysis had improved statistical powerfor exploring this association. Second, the protocol for thismeta-analysis, using explicit methods and criteria forstudy selection, data extraction, and data analysis, waswell designed before it was initiated. Third, a stringentsearching strategy based on computer-assisted and man-ual searches was applied to include as many eligiblestudies as possible. Finally, the quality of the studiesincluded in our meta-analysis was satisfactory; in fact,each article exhibited at least five stars. However, therewere still certain limitations of this meta-analysis. First, thesample sizes of several of the studies included in our meta-analysis were relatively small. Second, due to the limited

Table 3 - The results of the sensitivity analysis with each rs11196218A/G-focused study omitted.

Study omitted OR 95% CI p-value

Lin et al. 1.01 0.96-1.07 0.62Luo et al. 1.03 0.98-1.08 0.32Ma et al. 1.02 0.97-1.07 0.44Ng et al. 1.00 0.95-1.05 0.90Qiao et al. 1.02 0.97-1.07 0.41Wen et al. 1.03 0.98-1.09 0.26Zhai et al. 1.04 0.98-1.11 0.16Zhang, Y. 1.03 0.98-1.08 0.32Zheng et al. 1.02 0.97-1.07 0.50Zhu, H. 1.01 0.96-1.06 0.69

Abbreviations: OR, odds ratio; CI, confidence interval.

Figure 4 - Funnel plot analysis to detect publication bias (G vs. A of the rs11196218A/G polymorphism). Each point represents anindependent study on the indicated association. The dark point represents two overlapping articles.

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information on environment and lifestyle available theincluded studies, it was not possible to perform asubgroup meta-analysis or an interaction analysis basedon environment or lifestyle. Finally, similar to a case-control study, a meta-analysis is a retrospective study, andrecall bias might also exist.In conclusion, we did not find any association between the

TCF7L2 gene rs11196218A/G polymorphism and T2DM riskin the Chinese Han population. To achieve a better and morecomprehensive understanding of the association betweenTCF7L2 gene polymorphisms and T2DM risk, we suggestthat studies including large samples and different ethnicitiesand lifestyles and examining sophisticated gene-gene andgene-environment interactions should also be considered infuture analyses.


Ma ET and Wei L developed the idea for the study and drafted themanuscript. Ma ET, Wei L, Wang HL and Tian RR were responsible forconducting the search, the data collection and the study quality assessment.Ma ET, Wei L Wang HL and Guo J analyzed and interpreted the data. Allof the authors read and approved the final version of the manuscript.

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